JP2005517721A - Chemical sensitization by liposomes containing oligonucleotides. - Google Patents

Abstract

The present invention relates to the use of cationic liposome formulations containing oligonucleotides to increase the efficacy of chemotherapy and / or radiation therapy, and in particular, means for chemically sensitizing cancer or tumor tissue to the efficacy of chemotherapy. To the use of the liposomes as This is particularly advantageous in the context of treatment of ras expressing tumors such as breast cancer, lung cancer, pancreatic cancer, prostate cancer.

Description

RELATED APPLICATIONS This application is a continuation-in-part of US application Ser. No. 09 / 538,241, filed on Mar. 30, 2000, and US application Ser. No. 09 / 538,241 was issued on July 15, 1999. US application No. 09 / 354,109, which is a continuation-in-part of U.S. Application No. 09 / 354,109, which is then filed on Oct. 24, 1997. No. 08 / 957,327, which claims priority to provisional application No. 60 / 041,192, filed March 21, 1997. All of these applications are hereby incorporated by reference in their entirety.

Government Rights This study was supported by a grant from the National Institutes of Health. The United States government has certain rights in the invention.

FIELD OF THE INVENTION The present invention relates to a method of treatment using a cationic liposome composition containing an oligonucleotide or a combination of oligonucleotides that specifically binds to a gene expressed by tumor tissue, preferably chemotherapy or chemotherapy and radiation therapy. And new for sensitizing tumor tissue.

BACKGROUND OF THE INVENTION The use of chemotherapeutic agents for the treatment of cancer is well established. Examples of chemotherapeutic agents that have found established application in the treatment of cancer include, for example, tamoxifen, cisplatin, methotrexate, adriamycin, to name a few. Often such chemotherapeutic agents are used in combination, i.e., as a co-tail of a chemotherapy regimen, and in combination with other types of treatments such as radiation, surgery, or antibody-based therapies. Is done.
Although chemotherapeutic agents have successfully treated many different types of cancer, such as some leukemias, breast cancers, and prostate cancers, chemotherapy is problematic. For example, chemotherapeutic agents are often only effective against a limited number of cancers. Many chemotherapeutic agents are also toxic to non-target tissues. For example, it can cause nephrotoxicity. Another common problem with chemotherapy is that tumor tissue can become resistant to certain chemotherapeutic agents. For example, some tumors are known to become resistant to cisplatin.

  To alleviate such problems, it is known to co-administer chemotherapeutic agents, thereby minimizing the risk of tumors becoming resistant to chemotherapeutic regimens. However, this solution is not satisfactory. It does not exclude the fact that some tumors should become resistant to chemotherapy. This is disadvantageous, for example, when it results in the effectiveness of such chemotherapeutic agents that need to be administered in large doses to induce cytotoxicity. This is a problem because it increases the risk of systemic toxicity to non-target tissues. Also, it (if) can greatly increase the cost of treatment.

  It is likewise known that tumors can become resistant to ionizing radiation. For example, tumor resistance can be associated with, inter alia, the expression of certain oncogenes such as ras, raf, cot, mos, myc, as well as growth factors such as PDGF, FGF.

  The use of oligonucleotides for the treatment of cancer has also been reported, particularly the use of antisense oligonucleotides that target oncogenes or other genes expressed by specific cancers. However, antisense therapy also has some problems that hinder its effectiveness, in particular such oligonucleotides are unstable in vivo and thus before reaching the target site, eg tumor cells or virus infected cells. Antisense therapy is susceptible to the fact that it can become degraded.

  Attempts to increase the potency of oligos have included the synthesis of several analogs with modifications primarily directed at the phosphodiester backbone. For example, phosphorothioate oligonucleotides have been shown to exhibit increased resistance to nuclease digestion. Other modifications to the oligonucleotide included lipophilic moieties such as cholesterol and derivatization with polylysine to increase cellular uptake. Alternatively, the polyanionic nature of the molecule was removed in the methylphosphonate analog.

Another reported approach has been the use of cationic liposomes to enhance delivery. Bennet et al., Mol. Pharmacol. 4: 1023-1103 (1992); Zelphati et al., J. Biol. Lipsome Res. 7 (1): 31-49 (1997); Thierry et al., Biochem. Biophys. Res. Comm. 190 (3): 952-960 (1993). Cationic liposomes have sufficient charge to neutralize negatively charged oligonucleotides to facilitate interaction with negatively charged cell surfaces, as well as provide sufficient residual positive charge to the complex. It is widely accepted that it must have. (Litzinger et al., J. Liposome Research, 7 (1): 51-61 (1997)). However, problems associated with conventional cationic liposome delivery systems also include serum instability, undesired biodistribution, target non-specificity, which can be used for efficient nucleic acid delivery in vivo. This has hindered its use.
Therefore, methods for improving the treatment of chemoresistant tumors would be very advantageous.

OBJECT AND SUMMARY OF THE INVENTION An object of the present invention is to sensitize tumor tissue to the effect of chemotherapy by administration of at least one oligonucleotide targeting a gene expressed by tumor tissue as an adjuvant for chemotherapy. It is.
A more specific object of the present invention is to sensitize tumor tissue to the effects of chemotherapy by administration of at least one antisense oligonucleotide contained in a serum stable cationic liposome delivery system, wherein The oligo-containing cationic liposomes can be administered before, during or after chemotherapy.
A more specific object of the present invention comprises, as a cationic liposome delivery system, to sensitize tumor tissue to the effects of chemotherapy, at least one oligonucleotide targeting a gene expressed by the tumor tissue, Use liposomes with increased serum stability and targeting ability, including dimethyldioctadecylammonium bromide (DDAB), phosphatidylcholine (PC), and cholesterol (CHOL).

Another specific object of the present invention is a cationic lipid that is embedded in at least one oligonucleotide directed to a gene expressed by tumor tissue as a chemosensitized cationic liposome delivery system. , 2-dimyristoyl-3-trimethylammoniumpropane (DMTAP); using cationic liposomes containing phosphatidylcholine (PC) and cholesterol (CHOL).
Another object of the present invention is to provide 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), N- (2,3- (dioleoyloxy) propyl) -N, as a chemically sensitized cationic delivery system. N, N-trimethylammonium chloride, or 1- [2- (9- (Z) -octadecenoyloxy) -ethyl] -2- (8- (Z) heptadecenyl) -3- (2-hydroxyethyl) -Using cationic liposomes comprising at least one cationic lipid selected from imidazolinium chloride), phosphatidylcholine (PC) and cholesterol (CHOL).

An even more specific object of the present invention is a cationic comprising a 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); phosphatidylcholine (PC) and cholesterol (CHOL) as a chemosensitized cationic delivery system. Liposomes, each having a molar ratio of 0.5 to 1.4; 2.0 to 4.0; 0.5 to 2.5, more preferably 0.75 to 1.25; 3.0 to 4.0; 1.0 to 2.0, optimally using cationic liposomes that also range from about 1: 3.2: 1.6 (also including at least one oligonucleotide).
Another specific object of the present invention is a cationic liposome containing dimethyldioctadecyl ammonium bromide (DDAB), phosphatidylcholine (PC) and cholesterol, each having a molar ratio of 0.5 to 1.5; 0-4.0; 0.5-2.5, more preferably 0.75-1.25; 3.0-4.0; 1.0-2.0, optimally about 1: 3. 2: To produce a chemosensitizing formulation composed of cationic liposomes that are 1.6 (also comprising at least one oligonucleotide that targets a gene expressed by the tumor).

  Another specific object of the present invention is as a vehicle for in vivo delivery of one or more oligonucleotides to sensitize tumors, particularly chemoresistant tumors, to the effects of a particular chemotherapeutic regimen. 2-Dimyristoyl-3-trimethylammoniumpropane (DMTAP), dimethyldioctadecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammoniumpropane, (DOTAP) N- [2,3- (dioleoyloxy ) Propyl) -N, N, N-trimethylammonium chloride and 1- [2- (9- (Z) -octadecenoyloxy) -ethyl] -2- (8 (Z) heptadensenyl) -3- ( At least one cationic lipid selected from 2-hydroxyethyl) imidazolium chlorine; Cationic liposome containing Lucolin and Cholesterol, wherein the molar ratio of total cationic lipid, phosphatidylcholine and cholesterol is preferably 0.5-1.5; 2.0-4.0; 0.5-2. 5, more preferably 0.75-1.25; 3.0-4.0; 1.0-2.0, optimally. Use cationic liposomes that range from 8 to 1.2; 3.0 to 3.5; 1.4 to 1.8. The oligonucleotide can be in sense or antisense orientation relative to a gene target expressed by the tumor tissue, eg, a tumor gene. Optimally, the oligonucleotide will be an antisense oligonucleotide, preferably an antisense oligonucleotide with serum in the range of about 8-100 nucleotides, more preferably 15-40 nucleotides.

An even more specific object of the present invention is to improve the effect of chemotherapy on head and neck cancer, prostate cancer, pancreatic cancer, breast cancer, lung cancer, kidney cancer, ovarian cancer, brain cancer, esophageal cancer, sarcoma, carcinoma, To chemically sensitize solid tumors and cancers, including myeloma, bladder cancer, liver cancer, colon cancer, penile cancer, B and T cell lymphoma, leukemia, testicular cancer, bone cancer, and other blood cancers, Using the subject cationic liposomes comprising at least one oligonucleotide.
Another specific object of the invention is to sensitize cancer cells to the effects of chemotherapy and also potentially radiation therapy, as an adjunct to chemotherapy and possibly radiation therapy, ras, raf, ot, mos, myc, preferably an antisense oligonucleotide corresponding to a portion of an oncogene or growth factor PDGF, FGF, EGF, preferably selected from the group consisting of c-raf-1. Preferably, such oligonucleotides will be administered using the cationic liposome delivery system of the subject subject matter described above. The base contained in the oligonucleotide may or may not be modified, and the size of such oligonucleotide is preferably 8-100 nucleotides; more preferably 12-60 nucleotides; optimally It will be in the range of 15-40 or 15-25 nucleotides.

An even more specific object of the present invention is to provide 5'-GTG-CTCCATTGATGC-3 'and / or 5'-CCTGTATGTGCCTCATT-GATGCAGC-3', preferably embedded in cationic liposomes as adjuvants for chemotherapy. Administering an oligonucleotide comprising: Another specific object of the present invention is that at least one oligonucleotide, preferably an antigen corresponding to a surface or internal antigen or oncogene expressed by a tumor, prior to, simultaneously with, or after administration of a chemotherapeutic agent. Chemosensitization of tumor tissue to chemotherapeutic agent (s) by administration of cationic liposomes containing sense oligonucleotides.
In a more specific embodiment, the cationic liposome delivery system comprises a raf antisense oligonucleotide, the cancer being treated is prostate cancer, and chemotherapy will be a regimen that is effective against prostate cancer.
In another specific embodiment of the invention, the oligonucleotide will be chemically modified (eg, phosphorothioated).

Detailed Description of the Invention The present invention relates to a cationic liposome formulation comprising at least one oligonucleotide targeting a gene expressed by a tumor, e.g., a solid tumor, in order to sensitize the tumor to the effect of a chemotherapy regimen. Regarding use. In previous applications (included herein by reference below), the inventors have shown that the novel cationic liposome delivery system makes tumor cells more sensitive to the effects of radiation therapy. This application is an extension of that discovery. This same cationic liposome delivery system has now been found to make tumor cells more sensitive to chemotherapy. Based on that, the present invention relates to the combination of phosphatidylcholine (PC) and cholesterol (CHOL), dimethyldioctadecylammonium bromide (DDAB), 1,2-dithiophene, in order to chemosensitize tumor tissue to the effects of chemotherapy. Myristoyl-3-trimethylammoniumpropane (DMTAP), N-2,3- (dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride, 1- [2- (9 (Z) -octadece Noyloxy) -ethyl] -2- (8 (Z) heptadecenyl) -3- (2-hydroxyethyl) -imidazolinium chloride, 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) Preferably composed of at least one cationic lipid Relates to the use of a liposomal liposome comprising at least one oligonucleotide. Preferably, the molar ratio of cationic lipid: phosphatidylcholine: cholesterol is 0.5-2.5: 2.0-4.0: 0.5-0.5-2.5, more preferably. 7-1.5: 2.5-3.5 and 1.0-2.0, optimally 0.8-1.2: 3.0-3.6 and 1.4-1.8 Will.

  A particularly preferred molar ratio of cationic lipid: phosphatidylcholine: cholesterol is about 1: 3.2: 1.6. The subject cationic liposomes can be prepared by known methods. Preferred methods are described in the examples below. In essence, such methods involve dissolving a lipid in a solvent (eg, chloroform or methanol), evaporating to dryness, hydration at low temperature, an active agent (eg, in phosphate buffered saline (PBS)). Oligonucleotide), vigorous vortexing and sonication, and removal of non-embedded oligos. These methods have been found to provide high capture efficiencies (over 90%). Oligos embedded by the resulting liposomes can be stored, for example, at 4 ° C. and then used. For example, in the case of oligos embedded in liposomes, the liposomes can be stored for at least about 2 weeks.

  The cationic liposomes of the present invention comprise the desired oligonucleotide, and optionally other active agents such as peptides or proteins (eg, antibodies, growth factors, cytokines, enzyme hormones, receptors or fragments), drugs or chemotherapy. Used to embed agents, radionuclides, oligosaccharides, fluorophores, or diagnostic agents (such as radionuclides, detectable enzymes or fluorophores). In a preferred embodiment, the subject cationic liposomes will be used to embed antisense oligonucleotides that target genes that are specifically expressed by tumors treated by chemotherapy. Even more preferably, the antisense oligonucleotide will bind to an oncogene such as raf.

  As shown in the Examples, the cationic liposomes of the present invention provide high embedding efficiency, protect oligonucleotides from degradation in plasma for extended periods of time (ie, over 8 hours), and deliver efficiently to target cells It has been found that it enhances the intracellular availability of intact oligonucleotides and enhances the effectiveness of chemotherapy and radiation therapy very effectively.

  Also, as shown in the examples herein, the subject oligos included for the cationic liposome delivery system are ionizing radiation and chemotherapy for tumor tissue, including tumors that are resistant to the effects of chemotherapy and radiation. To enhance the effect. While not wishing to be bound by that idea, the subject cationic delivery system delivers oligos very effectively to target tissues, i.e., tumors that express the genes that the oligos target, which is responsible for gene expression. The present invention has a hypothesis of inhibiting. Instead, this is thought to make these cells more sensitive to the effects of chemotherapy and / or radiation therapy. One hypothesis is that this has an effect on cells that make them more susceptible to apoptosis. Preferably, the oligo will contain a modified base to enhance in vivo stability. However, it involves the use of oligonucleotides with or without modified bases (eg modifications to the phosphorothioate backbone such as phosphorothioate modified oligos, lipophilic modified oligos (eg cholesterol or poly-L-lysine)).

Preferably, the oligonucleotide will be an antisense oligonucleotide corresponding to a part of an oncogene or viral gene such as raf, ras, cot, mos, myc, myb, erb-2 and the onset and progression of cancer Try to inhibit the expression of genes involved in. In a preferred embodiment, oligonucleotides embedded with these liposomes will be used to treat cancer resistant to chemotherapy and possibly radiation therapy. It should be noted that cationic liposomes can also contain active agents other than oligonucleotides if the incorporation of active agents other than oligonucleotides has no deleterious effect on the oligo / cationic delivery system. This is considered because it is well established to treat cancer with a variety of different treatment regimens. Alternatively, if another active agent is utilized, it can be administered in naked form or using a different delivery system.
As found, oligo / cationic delivery can be administered before, contemporaneously and / or after chemotherapy or radiation.

The subject liposomes are also peptides, such as haptenic peptides, proteins, antibody hormones, growth factors and fragments thereof, chemotherapeutic agents, radionuclides, oligosaccharides, lectins, receptors, cytokines or monokines, antitumor agents, and others Can be used to embed various active agents. Examples include interleukin, interferon (α, β, γ), colony stimulating factor, tumor necrosis factor, methotrexate, cisplatin, doxorubicin, daonorubicin, fibroblast growth factor, and platelet-derived growth factor. .
Examples of radionuclides include radioactive species of yttrium, indium, and iodine.

The amount of active agent embedded in the subject liposomes will be at most that amount that maintains liposome stability after embedding. In the case of oligonucleotides, the amount of oligonucleotide is in the range of about 0.1 to 1,000 μg oligo / lipid mg, more preferably about 1 to 100 μg oligo / lipid mg, optimally about 10 to 50 μg oligo / lipid mg. I will.
However, this amount can vary for different active agents. The subject liposomes will be administered in combination with a pharmaceutically acceptable carrier such as glucose or saline phosphate buffered saline. Liposomes can also contain preservatives, emulsifiers, or surfactants often used in pharmaceutical formulation.

  The subject active agent-containing liposomes can be administered in different ways. Systemic and non-systemic methods of administration are appropriate. Such methods include injection (intramuscular, intraarterial, intraperitoneal, intravenous, intratumoral or other site specific injection, intrathecal, inhalatories, oral administration and topical methods. Preferred methods include intratumoral and intravenous methods of administration.

  The effective dosage will depend on the embedded drug, the disease or condition being treated, the patient being treated, other treatments, and other known factors. For oligonucleotides, the local dosage will be in the range of about 0.1 to about 500 μg. Typically, an amount that results in a serum concentration of oligo or other drug in the range of about 0.1 μg to 1000 μg / ml will be administered.

As discussed, surprisingly, when used as an adjunct to radiation therapy, chemotherapy, oligonucleotides, such as antisense oligonucleotides, have been found to enhance the effectiveness of radiation therapy and chemotherapy. It was. For example, antisense oligonucleotides corresponding to oncogenes such as raf can be used to increase the sensitivity of tumor cells to chemotherapy and radiation therapy, thereby increasing efficacy. While not wishing to be bound by this, it is theorized that such oligonucleotides can make tumor cells more susceptible to lysis or apoptosis (programmed cell death).
Specifically, this was demonstrated with raf antisense oligonucleotides. In this embodiment of the invention, antisense oligonucleotides corresponding to oncogenes such as raf will preferably be administered in liposome-embedded form before, simultaneously with, or immediately after chemotherapy and / or ionizing radiation therapy.

In the combinations of the invention involving radiation therapy, irradiation will be done by known methods, eg, using [M _cs ] irradiation, or other suitable device that delivers the radiation to the tumor site. The dose will be sufficient to cause tumor regression or remission. As demonstrated by the results, the combination of antisense oligonucleotides and radiation or chemotherapy was found to have a synergistic effect on tumor remission, especially on radiation resistant tumors. Therefore, the present invention may allow the use of lower doses of radiation or chemotherapy than previous therapies. However, of course, the amount of radiation or chemotherapy will depend on factors including patient condition, weight, other therapies and the like. Selection of appropriate radiation dosages and treatment regimens is well within the scope of those skilled in the art.

  The size of the administered oligonucleotide will preferably be 100 nucleotides or less, more preferably 40 nucleotides or less, or about 8-40 nucleotides, more preferably about 15-40 nucleotides, or 15-25 nucleotides. The size of the antisense oligonucleotide is such that, upon in vivo administration, the anti-tumor effect is produced by disrupting genes whose expression is involved in tumor growth, metastasis or apoptosis, or genes that sensitize tumor cells to radiation therapy. It is a thing.

  The present invention encompasses the use of the subject oligo / cationic liposome delivery system with any chemotherapy or combination of chemotherapy (the effectiveness of the chemotherapy or chemotherapy combination is thereby increased). Preferably, the oligo / cationic liposome delivery system will be used to treat tumors that are resistant to chemotherapy, for example as a result of prolonged administration of certain chemotherapeutic agents.

  In particular, as shown in the Examples, the administration of several oligonucleotides contained in the cationic liposome formulation of the present invention demonstrates the effectiveness of many different chemotherapeutic agents in mouse tumor models of human prostate cancer and human pancreatic cancer. Increasing was observed in several tumor models. Increased efficacy has been demonstrated on the basis of decreased tumor volume and inhibition of tumor growth for animals treated with liposomal compositions alone or chemotherapeutic agents alone, and particularly relative to controls. This has been shown for several different chemotherapeutic agents, epirubicin, mitoxantrone, cisplatin, gemzar, and gencitabine HCl. These results are included in FIGS.

Based on these results, the subject oligo-containing cationic liposome compositions are exemplified by alkylating agents, antimetabolites, apoptosis inducers, platinum coordination complexes, natural products, hormones, hormone antagonists, receptor agonists and receptor antagonists, anthracene In addition to diones, substituted urea methylhydrazine derivatives, adrenocortical inhibitors, small molecule inhibitors, peptides, antibodies and antibody fragments, enzyme inhibitors and tyrosine kinase inhibitors, etc. alone or in combination It is anticipated that it can be used as a means to enhance other chemotherapeutic agents.
Specific examples of such chemotherapeutic agents include doxorubicin, daunorubicin, methotrexate, adriamycin, tamoxifen, toremifene, cisplatin, epirubicin, docetaxal, paclitatol, gemzar, gencitabicin HCl, myxotantron, and others useful for the treatment of cancer And known chemotherapeutic agents.

  Examples of cancers for which the claimed combination therapy is useful include solid tumors and non-solid tumors, including those that have metastasized. Therapies can be used for any stage of cancer, ranging from precancerous lesions to advanced stage cancer. Specific examples include prostate cancer, pancreatic cancer, breast cancer, B and T cell leukemia, lymphoma, bone cancer, head and neck cancer, stomach cancer, bladder cancer, esophageal cancer, lung cancer (eg, large cell, small cell). Ovarian cancer, testicular cancer, myeloma, sarcoma, carcinoma, brain cancer, and others.

  In a preferred embodiment, the cancer to be treated includes a raf-expressing tumor such as human pancreatic cancer or human prostate cancer, and the chemotherapeutic agent includes cisplatin, myxotantron, epirubicin, gencitabicin, or gemzar. .

  The amount and regimen of chemotherapeutic agent administered will generally be as usual for a particular chemotherapeutic agent when administered alone or in combination with other chemotherapeutic agents. For example, such dosages range from about 0.00001 g / kg body weight to about 1-5 g / kg body weight, depending on the particular chemotherapeutic agent (and if combined with other therapies). It can be. The chemotherapeutic agent will be administered before, simultaneously with, or after administration of the oligo / cationic liposome composition of the present invention. Preferably, the chemotherapeutic agent will be administered after the liposomal composition. It is theorized that the subject cationic compositions make tumor cells more susceptible to apoptosis. However, we do not want to be bound by that idea.

Cationic liposome compositions and chemotherapeutic agents will typically be administered parenterally, for example, subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally or by intrathecal injection. The composition will generally include a carrier or excipient, such as buffered saline.
The chemotherapeutic agent can also be used in combination with other therapies, such as, among other things, radiation, radioimmunotherapy (RIT), therapeutic enzymes, hormone therapy, surgery, among others.

Example 1
Materials and Methods Oligodeoxyribonucleotides Oligodeoxyribonucleotide sequences directed to the translation start site of human c-rnf-1 cDNA were analyzed using standard Labs Limited (Gaithersburg, MD, USA). The sense (ATG-S) and antisense (ATG-AS) raf ODN sequences were 5′-GCAT-CAATGGAGCAC-3 ′ and 5′-GTG-CTCCATTGATGC-3 ′, respectively. One terminal base bond at each end was modified to a phosphorothioate group using 3H-1,2-benzo-dithiol-3-1,1,1-dioxide as a sulfurizing agent. Oligos were synthesized on a 15 μm scale and purified on a reverse phase chromatography column. For quality control, small aliquots of each oligo preparation were labeled with ³² P end, visualized with polyacrylamide gel electrophoresis (20% acrylamide and 5% bis), then densitometric scan of the labeled product.

  For the synthesis of 5'-fluorescein labeled ATG-AS / S raf ODN, the 3 'and 5' base linkages were modified to phosphorothioate groups as described above. Fluorescein phosphoramidite (1-dimethoxytriyloxy-2- (N-thiourea- (di-O-pivaloyl-fluorescein) -4-aminobutylpropyl-3-O- (2-cyanoethyl)-(N, N -Diisopropyl) -phosphoro-amidite) was ligated to the 5 'end during the last three synthesis cycles, 0.25 ml of fluorescein amidite 0.1M solution in acetonitrile and tetrazole 0.45M solution in acetonitrile 0. Consisting of 25 ml co-addition and 15 min incubation After synthesis, the ODN was deprotected and cleaved from the support in 1.0 ml 30% ammonium hydroxide for 24 hours at room temperature. The fluorescein label was synthesized using fluorescein isothiocyanate (FITC) The same structure was modified with. Purification was performed using standard reverse phase chromatography cartridge. Purification ODN was eluted from the cartridge with 20% acetonitrile 1.0 ml, dried and resuspended in water.

Production of Cationic Liposomes Cationic lipids 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dimethyldioctadecylammonium bromide (DDAB) , Purchased from Avanti Polar Lipids (Alabaster, AL, USA). Blank liposomes were prepared using one of three cationic lipids, phosphatidylcholine (PC), cholesterol (CHOL) at a molar ratio of 1: 3.2: 1.6. LE-ATG-S and LE-ATG-AS raf ODN were prepared using DDAB, PC and CHO in a molar ratio of 1: 3.2: 1.6. In summary, lipids dissolved in chloroform or methanol were evaporated to dryness in a round bottom flask using a rotary vacuum evaporator. The dried lipid film was hydrated overnight at 4 ° C. by adding 1 ml of 1.0 mg / ml ODN in phosphate buffered saline (PBS). The film was dispersed by vigorous vortexing and the liposome suspension was sonicated for 5 minutes in a bath sonicator (Laboratory Supplements, Hicksville, NY, USA). The ratio of ODN to lipid was 30 μg ODN / mg lipid. Non-embedded ODN was removed by washing the liposomes and centrifuging 3 times at 25000 g for 30 minutes in PBS. ODN embedding efficiency was determined by scintillation counting of aliquots of the preparation (a small amount of ³² P-end labeled ODN was added to excess unlabeled ODN in the preparation). ODNs embedded in liposomes were stored at 4 ° C. and used within 2 weeks of production. Blank liposomes were prepared exactly as described above in the absence of ODN.

Cell Culture SQ-20B tumor cells were established from laryngeal squamous cell carcinomas of patients who had failed the entire course of radiation therapy. ⁴³ tumor cells were Dulbecco's modified Eagle medium supplemented with 20% heat inactivated fetal bovine serum (FBS), 2 mM glutamine, 0.1 mM non-essential amino acids, 0.4 μg / ml hydrocortisone, 100 μg / ml streptomycin, 100 U / ml penicillin. (GIBCO BRL, Grand Island, NY, USA).

Intracellular raf ODN uptake and stability assay Logarithmically growing SQ-20B cells were seeded in 6-well plates (1 × 10 ⁶ cells per well) in medium containing 20% FBS. The next day, the cells are switched to medium containing 1% FBS and incubated at 37 ° C. with 10 μM ³² P-labeled LE-ATG-AS raf ODN or ATG-AS raf ODN (1 × 10 ⁶ c.p.m./ml). did. After various intervals of incubation, cells were washed with PBS, trypsinized, and centrifuged. The cell pellet was rinsed twice with PBS, resuspended in 0.2M glycine (pH 2.8) and then washed again with PBS. This treatment stripped the membrane-bound ODN and interpreted residual radioactivity as a representative value of intracellular levels of ODN. The cell pellet was then dissolved in 1% SDS and intracellular radioactivity was determined by liquid scintillation counting.
For ODN stability studies, cells were seeded and 10 μM ³² P-labeled LE-ATG-AS raf ODN or ATG-AS raf ODN (1 × 10 ⁶ c.p.) in 1% FBS-containing medium at 37 ° C. for 4 hours. M./ml). After this initial incubation with ODN, the cells were washed 3 times with PBS and switched to medium containing 20% FBS. Incubation was continued for various times, then trypsinized and washed with PBS. The cell pellet was dissolved in 10 mM Tris-HCl, 200 mM NaCl, 1% SDS, 200 μg / ml proteinase K, pH 7.4 at 37 ° C. for 2 n. ODN was extracted with phenol: chloroform, water fractions were collected, and aliquots were analyzed by scintillation counting. Samples were normalized for equal radioactivity to correct for possible ODN efflux over time, followed by 15% polyacrylamide / 7M urea gel, autoradiography.

Study of pharmacological trends of raf ODN Male Balb / c nu / nu mice (Charles River, Raleigh, NC, USA; 10-12 weeks old) are maintained at the Research Resources Facility at Georgetown University according to approved methods. And freely fed plina feed and water. Mice were injected intravenously from the tail vein with 30 mg / kg of LE-ATG-AS raf ODN or ATG-AS raf ODN. 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours after injection, from one animal in each group under anesthesia, heparinized tube from the retroorbital sinus Blood was drawn and killed by cervical dislocation. The blood was immediately centrifuged at 300 g at 4 ° C. for 10 minutes to separate the plasma. The liver, kidney, spleen, heart, and lungs were quickly removed and rinsed with ice-cold normal saline. Plasma and organs were stored at -70 ° C until further analysis.
Antisense raf ODN was isolated from plasma samples using the phenol-chloroform extraction method and from tissues using the DNA extraction kit (Stratagene, La Jolla, Calif., USA). A raf ODN concentration standard was prepared by adding a known amount of ATG-AS raf ODN into blank plasma or blank tissue sample, followed by extraction as described above. The extract was loaded onto a 20% polyacrylamide / 3M urea gel and electrophoresed in TBE buffer. Gels were electroblotted onto nylon membranes in 0.5 times TBE buffer at 20 V for 1 hour and blots were analyzed overnight with ³² P-labeled sense probe (ATG-S) in Quickhyb buffer (Stratagene) at 30 ° C. raf ODN). The radiolabeled probe was labeled on the 5 'end of ATG-S'raf ODN with γ- ^32- P-ATP using T4 polynucleotide kinase and on a Chroma Spin-10 column (Clontech, Palo Alto, CA, USA). It was produced by purification. A 10-50 fold excess of probe was used to ensure saturation of all bands. Using a computer program (ImageQuant software, Molecular Dynamics, Sunnyvale, CA, USA), autoradiographs were scanned and the amount of ATG-AS raf ODN in various samples was calculated relative to a standard.

In vivo delivery of S / AS raf ODN Logarithmically growing SQ-20B cells were injected subcutaneously into the flank region on both sides of male Balb / c nu / nu mice under mild anesthesia (2 × 10 ⁶ cells). Prior to the start of ODN treatment, tumors were grown to an average tumor volume of 115 mm ³ . Two treatment routes were followed: intravenous and intratumoral. For intravenous delivery, mice were randomly divided into 6 groups. Three mice in each group were injected intravenously by bolus injection via the tail vein at a dose of 6 mg / kg per day for 5 days, LE-ATG-AS, LE-ATG-S, ATG-AS, ATG -S, blank liposomes, or normal saline were received. Mice were sacrificed 24 hours after the last treatment, organs and tumor tissues were quickly excised, rinsed with ice-cold normal saline and stored at -70 ° C.
For intratumoral delivery, mice were randomly divided into 3 groups. Three mice in one group received intratumoral injections of 4 mg / kg LE-ATG-AS raf ODN on the right flank and LE-ATG-S raf ODN on the left flank daily for 7 days. Two control groups (3 mice per group) received normal saline or blank liposomes. Mice were sacrificed 24 hours after the last treatment, tumor tissue was excised, quickly rinsed with ice-cold normal saline and stored at -70 ° C.

Raf-1 immunoprecipitation and immunoblotting assay For in vitro experiments, logarithmically growing SQ-20B cells were treated with LE-ATG-AS raf ODN, LE- at various doses and time intervals in medium containing 1% FBS. Exposure to ATG-S raf ODN or blank liposomes. After incubation, the cells were lysed in a buffer containing 500 mM Hepes (pH 7.2), 1% NP-40, 10% glycerol, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 μg / ml leupeptin. The lysate was clarified by centrifugation at 16000 g for 20 minutes and the protein concentration was determined (Pierce, Rockford, IL, USA). Whole cell lysates normalized for protein content were used with a rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) against the 12 carboxy terminal amino acids of human Raf-1 p74 conjugated with protein A-agarose. And used for immunoprecipitation of Raf-1. The immunoprecipitate was washed sequentially with cell lysis buffer, 0.5 M LiCl 100 mM Tris-HCl, pH 7.4 and 10 mM Tris-HCl, pH 7.4. Immune complexes are boiled in Laemmli sample buffer, separated by 7.5% SDS-PAGE, then immunoblotted with polyclonal anti-Raf-1 antibody, manufacturer's protocol (Amersham Corporation, Arlington Heights, IL, USA). ) To detect Raf-1 using an ECL reagent. Raf-1 protein expression was quantified using a computer software program (Image-Quant; Molecular Dynamics, USA).
For in vivo expression studies, tumor tissues and organs were homogenized in cell lysis buffer using a Polytron homogenizer (Westbury, NY, USA). Raf-1 expression was analyzed on tissue homogenates by immunoprecipitation and immunoblotting as described above.

Raf-1 protein kinase activity assay Logarithmically growing SQ-20B cells were treated with 10 μM LE-ATG-AS raf ODN, LE-ATG-S raf ODN or blank liposomes in 1% FBS-containing medium for 8 hours. Cells were lysed as described above and lysates normalized for protein content were immunoprecipitated with agarose-conjugated anti-Raf-1 antibody overnight at 4 ° C. Raf-1 phosphotransferase activity contains ²¹ , 30 mM Hepes (pH 7.4), 1 mM manganese chloride, 1 mM DTT, 0.1 mM ATP and 20 μCi [λ- ³² P] ATP (6000 Ci / mmol) as described above. In vitro assay was performed using its physiological substrate, mitogen activated protein kinase (MKK1) ³² , in kinase buffer. Radiolabeled reaction products were separated by 10% SDS-PAGE and autoradiographed. The MKK1 band was quantified using the Image-Quant program (Molecular Dynamics).

Radiation Survival Dose Response Assay An appropriate number of SQ-20B cells are seeded in duplicate T-25 tissue culture flasks (Corning, NY, USA) in 20% FBS-containing medium and allowed to adhere for 8 hours at 37 ° C. It was. The medium was replaced with medium containing 1% FBS and the cells were exposed to 10 μM LE-ATG-AS raf ODN, 10 μM LE-ATG-S raf ODN or 10 μM blank liposomes for 6 hours prior to irradiation. Irradiation was performed using a ¹³⁷ C _S gamma irradiator (JL Shepard MARK I irradiator) and a dose rate of 114 cGy / min. Cells were irradiated with a total dose of 1 Gy, 3 Gy, 5 Gy and 13 Gy and then incubated for 2 hours. The media in all flasks was then replaced with media containing 20% FBS and incubation was continued for 7-10 days. Viable colonies were fixed and stained with 0.5% methylene blue in methanol and 0.13% fuchsin carboxylic acid. Colonies containing more than 50 cells were scored and the data fit to a computer generated, single hit multiple target and linear-quadratic model of radiation survival response ⁴⁴ .

Experimental Results raf ODN's PC / CHOL / DDAB liposome formulation is non-toxic Cationic liposomes, as described in Materials and Methods, have three molar ratios of 1: 3.2: 1.5 (DDAB, DOTAP and DMTAP), phosphatidylcholine (PC), and cholesterol (CHOL) in combination. Initially, the amount of radiolabeled antisense (ATG-AS) or sense (ATG-S) raf ODN was added to the initial ODN by scintillation counting of aliquots of the preparation to determine the ODN capture efficiency of the liposomes. Asked. The PC / CHOL / DDAB formulation gave the greatest ODN capture efficiency (> 90%, n = 10). PC / CHOL / DOTAP and PC / CHOL / DMTAP liposomes were found to be very cytotoxic. Therefore, the following experiments were performed using liposomal PC / CHOL / DDAB formulations.
Fluorescein image analysis using fluorescein-labeled ATG-AS raf ODN was performed to visualize the embedding of ODN in liposomes. A heterogeneous sized population of liposomes was obtained, including relatively large (Figure 7) and small liposomes (less than 2 microns, data not shown). In general, liposomes showed a tendency to form small aggregates, which could be easily dispersed by gentle shaking. ODN appeared to be distributed in the lipid bilayer and aqueous space. We next compared the effects of blank liposome (BL), liposome-embedded antisense (LE-ATG-AS) and sense (LE-ATG-S) raf ODN on cell survival. Blank liposomes at concentrations corresponding to less than 10.0 μM LE-ATG-AS / S raf ODN were non-toxic as measured by clonogenic survival and trypan blue dye exclusion (data not shown). However, blank liposomes showed cytotoxicity at doses greater than 20 μM and therefore doses below 10 μM were used in the following in vitro studies. For in vivo studies, mice were treated intravenously (iv) with a daily dose of 6 mg / kg blank liposomes for 2 weeks and then monitored for the next 30 days. There were no signs of weight loss or discomfort, indicating that the liposome formulation was non-toxic in vivo.

Liposome embedding enhances raf ODN uptake in vitro and in vitro and in vivo stability When less than 2% of free ATG-AS raf ODN is exposed to 100 μM concentration in the presence of low serum (1%) We have previously shown that uptake by SQ-20B cells at 6 hours and maximum uptake is about 4% at 12 hours after treatment. In this study, we asked if liposome embedding would enhance ODN uptake and stability in tumor cells. The kinetics of cellular uptake of LE-ATG-AS raf ODN in the presence of 1% serum is shown in FIG. The intracellular level of ODN increased with time and reached a plateau 8 hours after incubation. Approximately 13% of all applied LE-ATG-AS raf ODN (10 μM) was taken up by the cells. These results indicate that tumor cells achieved a significant increase in intracellular levels of ODN when treated with a 10-fold lower concentration of LE-ATG-AS raf ODN compared to free ATG-AS raf ODN. ing.
To test the intracellular stability of LE-ATG-AS raf ODN, ³² P-labeled ODN were treated at various times after the initial treatment of cells with 4 hours radiolabeled LE-ATG-AS raf ODN (10 μM). It was collected. The integrity of the ODN was determined by denaturing gel electrophoresis as described in Materials and Methods. An intact raf ODN (15 mer) was identified and no degradation was observed until 24 hours (FIG. 8B Igne). In contrast, cells treated with equimolar concentrations of free ATG-AS raf ODN did not show detectable ODN at all time points (data not shown). In other studies, the 15-mer ODN was intact after 24 hours of incubation of LE-ATG-AS raf ODN in medium containing relatively high levels of serum (15% FBS) (data not shown) . These results suggest that liposome embedding protects raf ODN from serum nuclease-induced degradation.

The plasma pharmacokinetics of LE-ATG-AS raf ODN are shown in FIG. i. v. After administration, a peak plasma concentration of 6.39 μg / ml was achieved and was intact. ODN could be detected up to 24 hours. The decrease in the plasma concentration of LE-ATG-AS raf ODN follows a biexponential pattern, with an initial half-life (t ₁ / 2β) of 24.5 minutes and a final half-life t ₁ / 2β of 11.36 hours Met. The area under the plasma concentration-time curve of LE-ATG-AS raf ODN is 5.9 μg. Time / ml, systemic clearance was 75.94 ml / min / kg, and volume of distribution was 74.67 l / kg. In contrast, intact and free ATG-AS raf ODN could only be detected at 5 minutes and the plasma concentration was 9.75 μg / ml. These findings are consistent with in vitro findings, where free ATG-AS raf ODN is rapidly degraded in plasma, while LE-ATG-AS raf ODN is present in the circulation for up to 24 hours. Suggests that.
The tissue distribution of LE-ATG-AS raf ODN is shown in FIG. Intact ODN was detected in all organs examined up to 48 hours (FIG. 4a). After administration of free ATG-AS raf ODN, intact ODN was observed only in 5 minutes in various organs, followed by degradation products (less than 15 mer) (data not shown). Previous reports of the pharmacokinetic profile of fully phosphorothioated ODN (S-oligo) delivered without a carrier suggest that the liver and kidney are the preferred sites for ODN accumulation. Our data is consistent with these studies, but it remains possible that liposome delivery may have facilitated targeting of ODN to specific tissues including the liver and kidney. This finding suggests that raf ODN in which only the 3 ′ and 5 ′ base bonds are phosphorothioated is rapidly degraded, and liposome embedding is an effective approach to maintain ODN stability in various organs for at least 48 hours. It suggests that there is.

Liposome-embedded ATG-AS raf ODN inhibits Raf-a expression and activity in vitro.
Time course experiments reveal that maximal inhibition of Raf-1 protein expression (52.3 ± 5.7%, approximately 74 kDa) occurred 8 hours after incubation of cells with 10 μM LE-ATG-AS raf ODN. (FIG. 5a). The inhibitory effect of LE-ATG-AS raf ODN (AS) was observed up to 24 hours (45.6 ± 9.8%). Raf-1 protein levels are comparable in control untreated cells (C), blank liposome treated cells (BL), LE-ATG-S raf ODN treated cells (S) (FIG. 5a), LE-ATG-AS. Raf ODN was shown to specifically inhibit Raf-1 protein expression in SQ-20B cells. Dose-response studies show that 35.94 ± 16.8% and 52.3 ± 5.7% inhibition of Raf-1 expression is 8 h cells with 5 μM and 10 μM LE-ATG-AS raf ODN, respectively. It showed what happened after the treatment (Figure 5b).
Using the physiological substrate mitogen-activated protein kinase (MKK1), we tested the effect of LE-ATG-AS raf ODN on the enzymatic activity of Raf-1 protein kinase ³² . Raf-1 protein kinase activity was comparable in control, untreated cells, LE-ATG-S raf ODN treated cells (10 μM, 8 hours). Consistent with the level of inhibition of Raf-1 protein expression, the in vitro phosphotransferase activity of Raf-1 protein kinase was inhibited in LE-ATG-AS raf ODN treated cells compared to control cells (10 μM, 8 Time; 62.6 ± 9.0%) (FIG. 6).

Liposome-embedded ATG-AS raf ODN inhibits Raf-1 expression in vivo In Balb / c nu / nu mice, the endogenous level of Raf-1 expression is different in different normal tissues and the expression level is reduced In turn, it was found that lung>liver>spleen>heart> kidney (data not shown). Interestingly, the anti-Raf-1 antibody recognized two protein bands (about 74 kDa and about 55 kDa) only in the kidney. It is unknown whether the smaller fragment is a proteolytic product of Raf-1 in mouse kidney (FIG. 7). Mouse and human c-raf-1 cDNAs share a conserved nucleotide sequence in the translation initiation region (Leszek Woznowski, personal communication). Therefore, we tested the effect of ATG-AS / S raf ODN on Raf-1 expression in normal mouse tissues. Free ATG-AS raf ODN i. v. After administration (6 days / day for 5 days), no inhibition of Raf-1 expression was observed in normal tissues (data not shown). However, it is not LE-ATG-S raf ODN, i.e. of LE-ATG-AS raf ODN. v. By administration (6 mg / kg per day for 5 days) Raf-1 (51.6 ± 17.4%; n = 3) and kidney (42.2 ± 11.0%; n = 3) A significant inhibition of about 74 kDa) was induced (FIG. 7). LE-ATG-AS raf ODN-related Raf-1 inhibition did not occur in the heart and lung (n = 3, data not shown). These findings were consistent with the normal tissue trend profile of LE-ATG-AS raf ODN and showed a relatively high accumulation of ODN in the liver and kidney compared to the heart and lung (FIG. 4b). It is still unclear whether Raf-1 inhibition in the liver and kidney is associated with any appreciable toxicity to these organs.

  Surprisingly, i.e. according to LE-ATG-AS raf ODN. v. Treatment has a variable effect on Raf-1 expression in SQ-208 tumor xenografts, ranging from 37.6-57.6% compared to LE-ATG-S raf ODN treated tumor xenografts. Inhibition level (n = 3) (FIG. 7). We interpret this as due to differences in tumor vasculature in different xenografts that prevent ODN delivery to poorly perfused tumor sites. Intravenous treatment with free ATG-AS / S raf ODN, LE-ATG-S raf ODN (S), brand liposomes, or normal saline (C), compared to untreated controls, Raf- 1 had no effect on expression. i. v. We study the effect of intratumoral administration of LE-ATG-AS raf ODN or LE-ATG-S raf ODN on Raf-1 expression by the variation in the level of Raf-1 inhibition observed after treatment. It was unleashed. The results shown in FIG. 8 show significant inhibition of Raf-1 protein expression in SQ-20B tumor xenografts after intratumoral treatment with LE-ATF-AS raf ODN compared to LE-ATG-S raf ODN. Shown (60.3 ± 5.4%; 11-3). Taken together, these data indicate that LE-ATG-AS raf ODN inhibits Raf-1 expression in a sequence specific manner in vivo.

SQ-20B cells treated with liposome-embedded ATG-AS raf ODN are radiosensitive. The radiation survival dose response of SQ-20B cells exposed to LE-ATG-AS raf ODN, LE-ATG-S raf ODN, or blank liposomes is shown in Table 1. The plating efficiency of cells treated with LE-ATG-S / AS raf ODN or blank liposomes is comparable (Table 1). Single hits, multiple targets (target models) and linear-quadratic function models (LQ) are most commonly used to analyze the radiation survival of cells. The target model is based on parameters D ₀ and fl. D ₀ is the reciprocal of the end slope of the survival curve and fl is an extrapolation of this slope to the vertical axis. The higher the D ₀ value, the more resistant the cells are to radiation-induced cell killing. Another parameter _Dq is a measure of the shoulder of the survival curve when the end slope line crosses the horizontal axis. The LQ model has two main parameters: α, a linear component that characterizes the radiation response at low doses; β, a secondary component that characterizes the response at high doses. The higher the α value, the more sensitive the cells are to radiation. The model free parameter D is called the mean inactivation dose and indicates the area under the survival curve plotted in linear coordinates. Clonogenic cell survival data was computer fitted into a single hit multiple target and linear-secondary model of the radiation survival response. After treatment with LE-ATG-AS raf ODN, the significant decrease observed in the values of radiobiological parameters D, D ₀ and D ₀ in SQ-20B cells is due to the antisense sequence specificity of Raf-1 protein kinase Suggest a good link between physical inhibition and radiosensitivity. Based on the ratio of mean inactivation dose, the dose modification factor (DMF) for LE-ATG-AS raf ODN treatment (10 μm) was about 1.6. These data are significant. This is because a 10-fold higher concentration of free ATG-AS raf ODN is required to achieve an equivalent level of radiosensitization of SQ-20B cells (ATG-AS raf ODN, 100 μm; DMF approximately 1 .4)

Appropriate numbers of cells were seeded in duplicate T-25 flasks per dose in each experiment as described in Materials and Methods. The plating efficiency of cells of blank liposome treatment, LE-ATG raf ODN treatment, and LE-ATG-AS raf ODN treatment were 65-79%, 52-83% and 59-90%, respectively.
Composite values for the various parameters were obtained from three experiments performed using LE-ATG raf ODN treated cells and two experiments performed using blank liposome treated cells.

Analysis The above results showed that the cationic liposome formulation of the present invention (PC / CHOL / DDAB) has several advantages. Specifically, the PC / CHOL / DDAB liposome formulation was found to be non-toxic and gave high ODN embedding efficiency.
We have identified several in vivo parameters that indicate that these cationic liposomes are suitable vehicles for safely and effectively transporting antisense oligos. Based on fluorescence microscopy, it appears that the oligo can be trapped inside the lipid bilayer (FIG. 1). This finding extends the first report showing the embedding of plasmid DNA in lipid sheets or tubes. By measuring plasma and tissue levels simultaneously, liposome embedding of oligos protects these relatively small DNA fragments from degradation in plasma and facilitates their tissue accumulation. Shown (FIGS. 3 and 4). Circulating antisense raf oligos retained in vivo by liposomes are intact for at least 24 hours, but no free oligos could be detected after 5 minutes. These data show that phosphodiester oligos with only two terminal phosphorothioate linkages at the 3 ′ and 5 ′ ends are similar to unblocked phosphodiester oligos, and these oligos disappear rapidly from the blood and almost accumulate tissue Is consistent with previous reports showing that It is hypothesized that the use of PC with cholesterol in our liposome preparations may have facilitated long-term retention of the oligo in the circulation and the tissue tendency and stability of the oligo.

  Particle size has been shown to play a major role in liposome biodistribution and cell entry pathways. Large liposomes are predominantly distributed in the reticuloendothelial system and can be ignored in other tissues, while small liposomes localize to other organs. Furthermore, the clearance of multi-layer vesicles with non-uniform size distribution follows a two-phase pattern. Large liposomes have rapid clearance and small liposomes have a slow rate of clearance. Only limited information is available regarding the biodistribution of oligonucleotide-containing cationic liposomes. Litzinger and co-workers have previously reported that oligonucleotides complexed with cationic liposomes about 2.0 microns in diameter are temporarily taken up by the lungs and then rapidly distributed in the liver. Recent studies have shown that endocytosis is the primary route of oligonucleotide delivery via cationic liposomes. Our liposome formulations consisted of both large and small liposomes. Consistent with the above idea, we show that the clearance of LE-ATG-AS raf ODN follows a biphasic pattern and is preferentially distributed in the liver.

  Liposome-embedded antisense raf oligo was non-toxic and inhibited Raf-1 expression in vitro and in vivo in a sequence specific manner (FIGS. 5-8 and Table 1). It should be noted that the intravenous and intratumoral routes of LE-ATG-AS raf ODN administration resulted in significant inhibition of Raf-1 expression in SQ-20B tumor tissue, both systemic and local administration Suggests the potential applicability of this compound. Furthermore, tumor cells treated with liposome-embedded antisense raf oligos were radiosensitive compared to control cells (Table 1). More recently, Dr. Experiments to show the radiosensitizing effect of liposome-embedded antisense raf oligo (5132) in SQ-20B tumor xenograft model in collaboration with Brett Monia (ISIS Pharmaceuticals, Carlsbad, CA, USA) Has started. The in vivo data obtained so far in athymic mice is promising (Gokhale et al., Unpublished data). This result suggests that liposomal delivery of ATG-AS raf ODN in combination with radiation may be an effective gene targeting approach for the treatment of cancer, particularly cancer that is resistant to standard radiotherapy.

Example 2
Materials and Methods Cell culture SQ-20B tumor cells were treated with 20% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 0.1 mM non-essential amino acids, 0.4 μg / ml hydrocortisone, 100 μg / ml streptomycin, 100 U / ml penicillin. Grown as a monolayer in Dulbecco's Modified Eagle Medium (DMEM) supplemented with (GIBCO BRL, Grand Island, NY).

Oligodeoxyribonucleotide 20-mer phosphorothioate antisense ODN corresponding to the 3 ′ untranslated region (3′-UTR) of human c-raf-1 mRNA (ISIS 5132/5132: 5′-TCC-CGC-CTG-TGA-CAT- GCA-TT-3 ') and 7 base mismatches phosphorothioate antisense ODN (ISIS 10353/10353; 5'-TCC -CGC- GCA - CTT -GAT-GCA-TT-3') were designed previously described (Monia et al., 1996a, b) as synthesized. The 20mer phosphorothioate sense ODN (5'-ATT-GCA-TGT-CAC-AGG-CGG-GA-3 ') was prepared as previously described (Soldatenkov et al., 1997), Lofstrand Labs Limited (Gaithersburg, MD). Was synthesized.

Preparation of Cationic Liposomes ODN was prepared as described previously (Gokhale et al., 1997) using dimethyldioctadecylammonium bromide, phosphatidylcholine and cholesterol (Avanti Polar Lipids, Alabaster, AL) in a molar ratio of 1: 3, 2: 1. , 6 embedded in the cationic liposome produced. In summary, lipids dissolved in chloroform or methanol were evaporated to dryness in a round bottom flask using a rotary vacuum evaporator. The dried lipid film was hydrated overnight at 4 ° C. by adding 5132/10353 at 1.0 mg / ml in phosphate buffered saline (PBS). The film was dispersed by vigorous vortexing and the liposome suspension was sonicated for 5 minutes in a bath sonicator (Laboratory Supplements, Hicksville, NY). The ODN / lipid ratio was 30 μg ODN / mg lipid, and an embedding efficiency of more than 90% was obtained. ODN (LE-5132 / LE-10353) embedded in liposomes was stored at 4 ° C. and used within one week. Blank liposomes (BL) were prepared exactly as described in the absence of ODN.

Raf-1 immunoprecipitation and immunoblotting assay For in vitro expression studies, logarithmically growing SQ-20B cells on day 1 were treated with LE-5132, 5132, LE-10353, or BL in medium containing 1% FBS for 6 hours. Exposure to various concentrations. The cells were then washed with 20% FBS-containing medium, liposomes removed, and incubation with 20% FBS-containing medium was performed in the presence of 5132 in the LE-5132 and 5132 treatment groups and 10353 in the LE-10353 group. Continued overnight (18 hours) in the presence. On the second day, the cells were rinsed with fresh 30% FBS and then a second course of the treatment schedule was performed as on day 1 for an additional 24 hours. This method gave minimal exposure of LE-5132 cells (12 hours). The cells were then transformed into a buffer containing 500 mM HEPES (ph 7.2), 1% NP-40, 10% glycerol, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 μg / ml aprotinin, 20 μg / ml leupeptin. Dissolved in. The lysate was clarified by centrifugation at 16,000 g for 20 minutes and the protein concentration was determined (Pierce, Rockford, IL). Whole cell lysates normalized for protein content were analyzed using a rabbit polyclonal antibody against the 12 carboxy terminal amino acids of human Raf-1 p74 (Santa Cruz Biotechnology, Santa Cruz, Calif.) With protein A-agarose binding. Used for immunoprecipitation. The immunoprecipitate was washed successively with cell lysis buffer, 0.5 M LiCl, 100 mM Tris-HCl, pH 7.4, 10 mM Tris-HCl, pH 7.4. Immune complexes were boiled in Laemmli sample buffer and separated by 7.5% SDS-PAGE. Thereafter, immunoblotting with a polyclonal anti-Raf-1 antibody, and Raf-1 detection using ECL reagents based on the manufacturer's protocol (Amersham Corporation, Arlington Heights, IL). Raf-1 protein levels were quantified using the computer software program Image-Quant (Molecular Dynamics, Sunnyvale, Calif.).
For in vivo expression studies, tumor tissue was homogenized in cell lysis buffer using a Polytron homogenizer (Westbury, NY). Raf-1 expression was analyzed on tissue homogenates by immunoprecipitation and immunoblotting as described above.

In vitro clotting assay The clotting assay was performed using normal human plasma containing known concentrations of LE-5132 or 5132. Activated partial thromboplastin time (APTT), APTT reagent (rabbit brain sephaline extract with olgic acid activator) (Sigma Diagnostics, St. Louis, MO) added to plasma sample, then manufactured Measured after addition of calcium chloride to initiate clot formation according to vendor recommendations (Sigma Diagnostics).
The time required to form a visible clot was recorded manually.

Pharmacokinetic Studies Male Balb / c nu / nu mice (mics) (National Cancer Institute, Frederick, MD) are maintained according to approved methods at the Division of Comparative Medicine at the University of Georgetown and freely available in prina diet and water. Gave. Mice were given 30 mg / kg of LE-5132 or 5132 formulated in PBS via the tail vein i. v. Injected. At 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours after injection, blood is drawn from the animal under anesthesia from the retroorbital sinus to a heparinized tube, Killed by partial dislocation. The blood was immediately centrifuged at 300 g for 10 minutes at 4 ° C. to separate the plasma. The liver, spleen, kidney, heart and lung were quickly removed and rinsed with ice-cold normal saline. Plasma and organs were stored at -70 ° C until further analysis.
Antisense raf ODN concentrations in plasma and tissue samples were detected as previously described by the inventors (Gokhale et al., 1997). In summary, ODN was isolated from plasma samples using the phenol-chloroform extraction method and from tissues using the DNA extraction kit (Stratagene, La Jolla, Calif.). A raf ODN concentration standard was prepared by adding a known amount of 5132 in a blank plasma sample or blank tissue sample, followed by extraction as described above. The extract was loaded onto a 20% polyacrylamide / 8M urea gel and electrophoresed in TBE buffer. The gel, 1 hour, TBE buffer 0.5 times at 20V, nylon membranes in electrically blot The blot overnight, QuickHyb buffer at 30 ° C. in (Stratagene) ^[32 P] labeled sense raf ODN probe Probed with. A 10-50 fold excess of probe was used to ensure saturation of all bands. Using a computer program (Image-Quant software), the autoradiograph was scanned and the amount of antisense raf ODN in the sample was calculated relative to a standard.

In Vivo LE-5132 Treatment and Irradiation Method: Anti-Tumor Efficacy Study Design Log-proliferating SQ-20B cells were s.c. c. Was injected (2 × 10 ⁰ cells). Prior to initiation of treatment, tumors were allowed to grow to an average tumor volume of approximately 72-94 mm ³ . Tumor volume was determined from caliper measurements of the three principal axes (a, b, c) and calculated using abc / 2, which is an approximation of the elliptical volume (πabc / 6).

For each experiment, tumor-bearing mice were randomly divided into different treatment groups (5-7 per group). The ODN group of mice received LE-5132 i.d. at a dose of 6-10 mg / kg per day or every other day for a total of 12-18 days, or by both methods. v. I received it. Tumors IR group ^were irradiated with ^[137 _{C S]} irradiator (J.J.Shepard Mark I). Animal restraint and normal tissue shielding were performed in a hinged semi-cylindrical plastic chamber mounted after a special form of 2.5 cm thick lead shielding. Tumor-bearing hind limbs were mounted by taping the limbs onto a 1.6 mm Plexiglas platform that protrudes from the chamber hole and is exposed to radiation. TLD dosimetry was used to determine the average dose rate to the center of the tumor and to the mouse body in advance using experimental irradiation conditions and a custom-made tissue equivalent mouse model. Dose distribution was confirmed in several mice. Tumors were irradiated at a dose rate of 2.37 Gy / min at 3.8 Gy per day for up to 18 days. Systemic dose was less than 5% of tumor dose (0.19 Gy / day). None of the mice experienced gastrointestinal problems during the experiment. The combination treatment group of animals received LE-5132 on day 0, and received LE-5132 and IR on days 1-6, 8, 10, 12 at intervals of 3-4 hours. In addition, this group received only IR on days 7, 9, 11, 13-18. Of the two control groups, one received BL on the same dosing schedule as LE-3132 (BL) and the other was untreated (C). Tumor volume was monitored once or twice a week and 12-27 days after the last treatment.
Tumor volume was calculated as a percentage of the initial tumor volume (day 0, day 1 of administration) and the mean tumor volume ± SE was plotted. Analysis of variance (one-way ANOVA) was performed to determine the statistical significance of changes in tumor volume observed 12 days after the last treatment.

Histopathology Tumor tissue was excised from a representative treatment group 24 hours after the last treatment, fixed with 10% formalin, and paraffin sections were analyzed under a microscope after hematoxylin / cosin staining. Histological changes such as apoptotic cells containing fragmented chromatin were scored under a light microscope (American Optical Corporation, Buffalo, NY). Ten fields with about 100 cells per field were scored in each treatment group.

Experimental Results LE-5132 inhibits Raf-1 expression in vitro To establish antisense ODN sequence-specific inhibition of Raf-1 expression, 5132, LE-5132, 7 Treated with base mismatch LE-10353 (FIG. 9A). Previously, in vitro inhibition of Raf-1 by 5132 has been shown to require lipofectin (Monia et al., 1996a). Consistent with this, 5132 was found to be ineffective in cell culture (FIG. 9A, lanes 4 and 6). A significant reduction in the level of Raf-1 protein was observed in vitro with LE-5132 compared to 5132 treatment (FIG. 9B, 0.5 μM LE-5132 LE-5132, 71.3 ± 22.5% 1.0 μM LE-5132, 79.6% ± 16.7%). Liposome-embedded mismatch antisense ODN (LE-10353) showed Raf-1 expression comparable to untreated or BL-treated cells (FIG. 9A, lanes 7-9). Taken together, these data establish the antisense sequence-specific titer of LE-5132 in SQ-20B cells.

LE-5132 uses normal human plasma as a step towards the clinical application of cationic liposomes for safe and effective delivery of ODNs that do not affect clotting in vitro, using 5132 and LE-5132 for clotting time. The present inventors compared the effects. Addition of 5132 to normal human plasma resulted in a concentration-dependent extension of clotting time (FIG. 10). Approximately 95% and 197.5% increases in APTT were observed in vitro in the presence of 5132 of 100 μg / ml and 200 μg / ml, respectively, while only a slight increase in APTT was observed in LE-5132 (100 μg / ml , 13%; 200 μg / l, 14.5%). BL in the same concentration range showed no effect on APTT (data not shown).

Liposome embedding enhances 5132 pharmacokinetics LE-5132 or 5132 single i. v. Pharmacokinetic parameters were obtained after bolus administration. As shown in FIGS. 11A and B, intact ODN could be detected in plasma for at least 8 hours in both cases. The peak plasma concentrations at 5 minutes after ODN administration were 28.5 μg / ml and 13.5 μg / ml for LE-5132 and 5132, respectively. Decreased plasma concentrations of LE-5132 and 5132 follow the biexponential pattern, with initial distribution half-lives (t ₁ / 2β) of 34. And 21.6 minutes. The final half-lives (t ₁ / 2β) for LE-5132 and 5132 were 14.5 hours and 4.3 hours, respectively. As shown in Table 2, the area under the plasma concentration-time curve (AUC) is 5.8 times higher for LE-5132 compared to 5132 and the clearance rate of intact ODN is 5132 compared to LE-5132. it was high.

  The normal tissue distribution profiles of LE-5132 and 5132 are shown as a function of AUC in FIG. After either treatment, intact ODN could be detected in the examined organs up to 48 hours (data not shown). However, the tissue distribution of LE-5132 was different from 5132 which is a free phosphorothioated ODN. Compared to 5132 administration, significantly higher levels of intact ODN could be measured in the liver (18.4 fold) and spleen (31 fold) after LE-5132 administration. Slightly higher ODN levels were observed in other organs via liposomal delivery of ODN compared to free ODN (3-fold heart; 1.5-fold lung). Interestingly, the level of intact ODN in the kidney was low (0.77 fold) with LE-5132. Further studies conducted showed moderately higher ODN levels in SQ-20B tumor tissue after LE-5132 treatment (1.4 fold) compared to 5132 treatment.

LE-5132 Inhibits SQ-20B Tumor Growth As shown in FIG. 13, the anti-tumor effect of LE-5132 is observed within one week after the start of treatment, and the mean tumor volume is observed during the next course of treatment. It continued to decrease. On the last day of treatment (19 days), the LE-5132 and BL groups were 42.0% ± 5.5% and 290.6% ± 26.6% of the initial volume (day 0, 100%), respectively. It was. The tumor volume of the LE-5132 group reached the first volume within 6-10 days after the last dose. At all time points after treatment, significant differences in tumor volume were observed between the LE-5132 group and the BL group. The study ended at 30 days, at which time the average tumor volume in the BL group was approximately 3.4 times greater than that in the LE-5132 group.

  In other studies, we compared the antitumor efficacy of LE-5132 and 5132. LE-5132 or 5132 (10 mg / kg) was administered i.d. to tumor-bearing mice daily for the first 7 days. v. Followed by three additional doses every other day. Control groups received similar treatment with BL or were not treated at all (C). Tumor volume was monitored for a total of 35 days. The mean tumor volume on day 35 compared to day 0 (100%) was BL, 427.8% ± 32.5%; C, 405.3% ± 26.8%; 5132, 159.6% ± 10 0.05; LE-5132, 105.3% ± 6.3%. ANOVA was performed to determine the significance of the difference in mean tumor volume in various categories at 35 days. Tumor growth patterns were comparable in the BL and C control groups. The 5132 and LE-5132 groups showed significant antitumor activity against the BL and C groups (n = 5, p <0.0001). However, the LE-5132 group showed greater antitumor activity against 5132 (n = 5, p <0.001). These data are consistent with 5132 relatively increased plasma, normal tissue, and tumor levels in liposome-embedded form.

LE-5132 inhibits Raf-1 expression in vivo Relative Raf-1 protein levels were measured in SQ-20B tumor tissue of mice exposed to LE-5132 ± IR or BL (FIG. 14).

  In the LE-5132 group, Raf-1 expression was 35.5% ± 13.4% and 27.7% ± 13.3% compared to the BL group (100%) on days 7 and 14, respectively. I found it. Inhibition of Raf-1 expression was also observed in SQ-20B tumors in mice treated with a combination of LE-5132 and IR. IR treatment alone did not change Raf-1 expression compared to untreated or BL controls (FIG. 14).

LE = 5132 is a tumor radiosensitizer. SQ-20B cells were established from tumors after failure of radiotherapy, and LE-5132 treatment caused tumor growth inhibition during the course of treatment. We asked whether control of the growth of radiation resistant tumors can be achieved by a combination of LE-5132 and IR treatment. LE-5132 (10 mg / kg) was administered 10 times over 12 days (Day 0-12). This treatment caused tumor growth inhibition compared to control mice (untreated and BL) for up to 1 week after the last dose (19 days). This was followed by a steady increase in tumor volume (FIG. 15A). IR (3.8 Gy / day) was given daily for 18 days. In this group, a modest decrease in mean tumor volume was observed by day 26 (14.9% ± 7.1% of initial volume) (FIG. 7A). Thereafter, the tumor grew and reached its initial volume within the next 3-7 days. The combination of LE-5132 and IR treatment caused a significant decrease in mean tumor volume by day 26 (57.9% ± 8.0% of initial volume). By 30 days, the mean tumor volume compared to the initial volume (100%, day 0) was LE-5132, 122.5% ± 13.8%; IR, 113.8% ± 17.6%; LE-5132 + IR, 43.5% ± 2.9%; LB / untreated control, 370.8% ± 15.6% (FIG. 15). Both LE-5132 and IR groups showed significant tumor growth inhibition relative to BL and untreated groups (p <0.001). Statistical analysis showed that the difference in tumor volume was not significant in IR versus the LE-5132 group. Very importantly, the LE-5132 + IR group showed significantly greater antitumor activity (p <0.001) than the LE-5132, IR, BL, untreated groups (FIG. 15B).

The combination of LE-5132 and IR treatment causes a significant increase in apoptosis in vivo. Representative tumors of various treatment groups were excised 24 hours after the last treatment of histopathological examination. Both necrotic and apoptotic cells were seen in the LE-5132 or IR groups compared to the untreated group. In addition, some clonal regrowth of living cells containing intact nuclei was observed in the IR group (data not shown). The proportion of apoptotic cells containing highly fragmented nuclei was significantly greater in the LE-5132 + IR group compared to the single agent or untreated control group (FIG. 16). The relative ratio of apoptotic / live cells scored in the different groups was C, 0.06 LE-5132, 0.64; IR, 0.72; LE-5132 + IR, 2.46.

Discussion Antisense ODN therapeutics are a novel approach to increase the effectiveness of anti-cancer agents by sequence specific inhibition of proliferation or survival signals. In our knowledge, this report provides the first evidence of the effectiveness of a well-characterized antisense ODN as a radiosensitizer or chemical sensitizer in animal tumor models. SQ-20B tumor cells were established from laryngeal squamous cell carcinomas of patients who were unsuccessful throughout the course of radiation therapy. Radiation or antisense ODN treatment alone caused transient inhibition of SQ-20B tumor growth but not tumor regression, whereas the combination of antisense raf ODN and radiation treatment allowed at least 27 days after treatment. Sustained tumor regression occurred (FIGS. 13 and 15). These data support the role of Raf-1 in cell proliferation and survival and establish antisense rad ODN as a novel in vivo radiosensitizer.

  We found significant inhibition of Raf-1 protein expression after treatment of SQ-20B cells and tumors with LE-5132, suggesting that LE-5132 is a bioactive compound in vitro and in vivo ( 9 and 14). Since the 5132 sequence corresponds to the 3'UTR of human c-raf-1 which is not conserved in mice, inhibition of Raf-1 in normal mouse tissues could not be studied. Previous studies have shown that, among other potential toxic effects, PS-ODN treatment causes injury associated with a dose-dependent extension of clotting time. The effect of complementation and clotting of PS-ODN containing 5132 could be avoided by changing the dosing regimen. Our results indicate that 5132 (LE-5132) embedding liposomes prevents changes in clotting time (FIG. 10). Also, liposomal delivery of ODN can mitigate many of the other sequence-independent side effects of PS-ODN, including hematological changes and complement activation. A significant increase in the plasma concentration of 5132 liposome formulations and most tissue levels was observed compared to free 5132 (FIGS. 11 and 12 and Table 2). Consistent with this, the anti-tumor titer of LE-5132 was found to be significantly higher than that of 5132. Taken together, these data suggest that liposome embedding is a potent method of ODN transport in vivo.

  The mechanism by which inhibition of Raf-1 expression increases IR-induced cytotoxicity is unclear. The role of Raf-1 as an anti-apoptotic or survival factor has been demonstrated in growth factor-deficient hematopoietic cells and v-abl-transformed NIH / 3T3 cells (Troppmair et al., 1992; Wang et al., 1996, Weissinger et al., 1997). ). Some reports indicate that the balance between cell death and cell survival signals determines the fate of cells exposed to genotoxic or non-genotoxic stress. IR has been shown to activate various types of signal molecules including Raf-1 protein kinase, mitogen activated protein kinase (MAPK), transcription factors AP-1 and NK-κB (Kasid and Suy, (Reviewed in 1998). One possibility is that Raf-1 can have a protective role in irradiated cells. We observed increased levels of Bax protein, a pro-apoptotic member of the Bcl-2 family, in SQ-20B cells treated with IR or a combination of LE-5132 and IR (data not shown). Radiation-induced Bax expression correlated with apoptosis (Zhan et al., 1994). Furthermore, histopathological examination shows a significant percentage of tumor cells containing fragmented chromatin that is an indicator of apoptosis in the LE-5132 + IR treated group compared to the LE-5132, IR, or untreated control group. (FIG. 16). These data suggest that Raf-1 can help promote anti-apoptotic signaling pathways in irradiated cells. Inhibition of Raf-1 by antisense raf ODN will then produce a substantial effect of IR-responsive pro-apoptotic signals including reversal of tumor radiation resistance.

Example 3
Materials and Methods Preparation of DMTAP: PC: CHOL liposomes having a molar ratio of 1: 3.2: 1.6 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP): phosphatidylcholine (PC): cholesterol (CHOL) Liposomes with anti-tumor raf oligonucleotide (ATG-AS) embedded therein were produced using substantially the same method as previously described.

In vitro results A) Increased cellular uptake of antisense raf oligodeoxyribonucleotides embedded in liposomes composed of DMTAP: PC: CHOL
Dose-response uptake experiment: SQ-20B tumor cells were treated with radiolabeled ( ³² P-γATP) and liposome-embedded form (LE-ATG-AS) or free form (ATG-AS) indicated dose Incubated with a mixture of unlabeled antisense raf oligonucleotide (ATG-AS) (FIG. 17). The treatment lasted for 4 hours at 37 ° C. in medium containing 1% serum. After incubation, the cells were washed with phosphate buffered saline (PBS), detached with trypsin treatment, and collected by centrifugation. The cell pellet was washed twice with PBS and then the cells were lysed in 1% sodium dodecyl sulfate. Intracellular radioactivity, an indicator of the amount of ATG-AS taken up by cells, was measured by liquid scintillation counting. The data shown in FIG. 17 shows a significant increase in the cellular uptake of LE-ATG-AS at all doses tested compared to ATG-AS.

Time course uptake experiments: SQ-20B tumor cells were treated with radiolabeled ( ³² P-γATP) and 4 μM unlabeled antisense raf in liposome-embedded form (LE-ATG-AS) or free form (ATG-AS). Incubated with a mixture with oligonucleotide (ATG-AS). Treatment continued for the indicated time at 37 ° C. in medium containing 1% serum (FIG. 18). After incubation, the cells were washed with phosphate buffered saline (PBS), detached with trypsin treatment, and collected by centrifugation. The cell pellet was washed twice with PBS and then the cells were lysed in 1% sodium dodecyl sulfate. Intracellular radioactivity, an indicator of the amount of ATG-AS taken up by cells, was measured by liquid scintillation counting. The data shown in FIG. 18 shows that LE-ATG-AS at all time points tested (15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 16 hours, 24 hours) compared to ATG-AS. Shows a significant increase in the intracellular accumulation of.

B. Intracellular stability of antisense raf oligodeoxyribonucleotides embedded in liposomes composed of DMTAP: PC: CHOL
Stability test: SQ-20B tumor cells were treated with radiolabeled ( ³² P-γATP) and liposome-embedded form (LE-ATG-AS) (FIG. 19, lane 1) or free form (ATG-AS) (FIG. 19. Incubated with a mixture of 10 μM unlabeled antisense raf oligonucleotide (ATG-AS) in lane 2). The treatment lasted 4 hours at 37 ° C. in medium containing 1% serum. Immediately after incubation, cells were washed with phosphate buffered saline (PBS), detached with trypsinization, and collected by centrifugation. The cell pellet was washed twice with PBS and then the cells were lysed in 10 mM Tris-HCl, 200 mM NaCl, 1% SDS, 200 μg / ml proteinase K, pH 7.4 for 2 hours at 37 ° C. The oligo was extracted with phenol: chloroform and the water fraction was collected. Samples were normalized for equal radioactivity and analyzed by denaturing gel electrophoresis (15% polyacrylamide / 7M urea) followed by autoradiography. The data shown in FIG. 19 shows the intact ATG-AS oligonucleotide (lane 1) in cells treated with LE-ATG-AS and the degraded form of this oligo in cells treated with ATG-AS (lane 2). . These data suggest that embedding in DMTAP: PC: CHOL liposome formulation inhibits oligo degradation.

Stability experiments: SQ-20B tumor cells were treated with radiolabeled ( ³² P-γATP) and 10 μM unlabeled antisense raf oligonucleotide (ATG-AS) in liposome-embedded form (LE-ATG-AS). Incubated with the mixture. The treatment lasted 4 hours at 37 ° C. in medium containing 1% serum. After incubation, the cells were washed with phosphate buffered saline (PBS) and incubation was continued for an additional hour in medium containing 20% serum. Cells were detached with trypsin treatment and collected by centrifugation. The cell pellet was washed twice with PBS and then the cells were lysed in 10 mM Tris-HCl, 200 mM NaCl, 1% SDS, 200 μg / ml proteinase K, pH 7.4 for 2 hours at 37 ° C. The oligo was extracted with phenol: chloroform and the water fraction was collected. Samples were normalized for equal radioactivity and analyzed by denaturing gel electrophoresis (15% polyacrylamide / 7M urea) followed by autoradiography. The data shown in FIG. 20 shows intact ATG-AS oligonucleotide (lane 1) in cells treated with LE-ATG-AS. A radiolabeled control ATG-AS standard is shown in lane 2. These data further suggest that embedding the oligonucleotide in a DMTAP: PC: CHOL liposome formulation protects the oligo and increases the intracellular availability of the intact oligo.

In vivo results <br/> In vivo data
Safety study of liposomes composed of DMTAP: PC: CHOL in CD2F1 mice: To determine the safety of cationic liposomes composed of DMTAP: PC: CHOL, 0, 1, 3, 4, 6, 7 On days 9, 10, 12, 13, 15, 16 these liposomes were injected intravenously into male CD2F1 mice (n = 5, 5 mg / kg, 15 mg / kg, 25 mg / kg, 25 mg / kg oligo concentration). Equal total lipids). Two of the five animals were sacrificed on day 18 due to pathology and the rest of the animals were sacrificed on day 31. Group weights were monitored throughout the study. As shown in FIG. 21, all animals survived the treatment and showed weight gain before the scheduled termination of the study.

Safety of antisense raf oligodeoxyribonucleotide embedded in liposome composed of DMTAP: PC: CHOL : embedded in antisense raf oligo (LE-ATG-AS) composed of DMTAP: PC: CHOL In order to determine the safety of the existing cationic liposomes, on 0, 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16 days, liposomes containing antisense raf oligos were treated with male CD2F1. Mice were injected intravenously (n = 5, 5 mg / kg, 15 mg / kg, 25 mg / kg, 35 mg / kg oligo concentration). Two of the five animals were sacrificed on day 18 for pathological examination and the rest of the animals were sacrificed on day 31. Group weights were monitored throughout the study. As shown in FIG. 22, all animals survived the treatment and showed weight gain. These data show that the LE-ATG-AS composition composed of DMTAP, PC and CHO, in which the antisense raf oligodeoxyribonucleotide is embedded, is safe. Is shown.

Example 4
Use of Raf Antisense Vigo and Chemotherapy in a Human Prostate Cancer Model This example demonstrates the efficacy of Leraf Aon in combination with cisplatin in athymic nu / nu mice bearing human prostate cancer (PC-3) xenografts. Regarding tumor efficacy. Male athymic mice were inoculated subcutaneously (sc) with 5 × 10 ⁶ PC-3 cells in 0.2 ml phosphate buffered saline per animal. Tumor growth until tumor volume is 60 mm ³ 100 mm ^3, were twice monitored weekly. The animals were randomly divided into the above 5 treatment groups (n = 8). Mice were treated intravenously with the indicated doses via the tail vein and tumor size was monitored twice a week. Values shown are mean ± s. d. It is. As shown in FIG. 23, the oligo / cationic liposome formulation significantly potentiated the effectiveness of cisplatin as shown by tumor volume reduction compared to the control.

Example 5
Use of Raf antisense oligos with chemotherapeutic agents in several animal models of human prostate cancer The antitumor efficacy of LEraf AON in combination with epirubicin carries human prostate cancer (PC-3) xenografts Thymus-deficient nu / nu mice were evaluated. Male athymic mice were treated with 5 × 10 ⁶ PC-3 cells in 0.2 ml phosphate buffered saline per animal. c. Vaccinated. Tumor growth until tumor volume is 60 mm ³ 100 mm ^3, were twice monitored weekly. The animals were randomly divided into the above 5 treatment groups (n = 6). Mice were treated intravenously with the indicated doses via the tail vein and tumor size was monitored twice a week. Values shown are mean ± s. d. It is. As shown in FIG. 24, the oligo / cationic system again enhanced the efficacy of chemotherapy, particularly epirubicin chemotherapy.

Example 6
Use of Raf antisense oligos with anti-chemotherapeutic agents in animal models of human prostate cancer The antitumor efficacy of LEraf AON in combination with mitoxantrone carries human prostate cancer (PC-3) xenografts Thymic deficient nu / nu mice were studied. Male athymic mice were treated with 5 × 10 ⁶ PC-3 cells in 0.2 ml phosphate buffered saline per animal. c. Vaccinated. Tumor growth until tumor volume is 60 mm ³ 100 mm ^3, were twice monitored weekly. The animals were randomly divided into the above 5 treatment groups (n = 6). Mice were treated intravenously with the indicated dose via the tail vein and tumor size was monitored twice a week. Values shown are mean ± s. d. It is. As shown in FIG. 25, the effectiveness of the chemotherapeutic agent mitoxantrone in this example was enhanced again by the oligo / cationic formulation.

Example 7
Anti-tumor efficacy of a combination of liposome-entrapped raf antisense oligodeoxyribonucleotide (LErafAON) and Gemzar (NDC0002-7501-01, Gencitabine HCl, Eli Lilly and Company, Indianapolis, IN), human pancreatic cancer xenograft (Colo357) Were evaluated in athymic mice carrying Athymic mice were inoculated with approximately 1.0 × 10 ⁶ Colo357 human pancreatic cancer cells. The tumor ^volume, was not on the size of 50mm ³ ~100mm 3. Tumor-bearing mice were randomly divided into 5 treatment categories (n = 5). LErafAON formulations were prepared as previously described (Gokhale et al., Gene Therapy 4,1299-1299, 1997; and Gokhale et al., Manuscript in preparation)> anti-cancer drug Gemzar (200 mg vial) Purchased from a hospital oncology pharmacy and reconstituted in normal saline (12.5 mg / ml). LErafAON (25.0 mg / kg) or Gemzar (75.0 mg / kg) was injected intravenously on the days indicated. Tumor volume was measured twice a week and the tumor size (% initial) for the various treatment groups was plotted. As shown in this figure, in the combination treatment group (LErafAON + Gemzar) compared to the single agent treatment group (LErafAON and Gemzar) or the control group (NS normal saline, BL, blank liposome) Growth inhibition was observed. Values show mean ± s. d. It is. The results again show that the cationic / oligo system enhanced the antitumor efficacy of chemotherapeutic agents.

Example 8
Use of Antisense Raf Oligo and Chemotherapy in Human Pancreatic Cancer Animal Model Combination of Liposome Capture Raf Antisense Oligodeoxyribonucleotide (LErafAON) and Gemzar (NDC0002-7501-01, Gencitabine HCl, Eli Lilly and Company, Indianapolis, IN) Anti-tumor efficacy was evaluated in athymic mice carrying human pancreatic cancer xenografts (Aspc-1). Athymic mice were inoculated with approximately 2.5 × 10 ⁶ Aspc-1 human pancreatic cancer cells. The tumor ^volume, was not on the size of 50mm ³ ~100mm 3. Tumor-bearing mice were randomly divided into 5 treatment categories (n = 5). LErafAON formulations were prepared as previously described (Gokhale et al., Gene Therapy 4,1299-1299, 1997; and Gokhale et al., Manuscript in preparation). The anticancer drug Gemzar (200 mg vial) was purchased from the Oncology Pharmacy at Georgetown University Hospital and reconstituted in normal saline (12.5 mg / ml). LErafAON (25.0 mg / kg) or Gemzar (100.0 mg / kg) was injected intravenously on the days indicated. Tumor volume was measured twice a week and the tumor size (% initial) for the various treatment groups was plotted. As shown in this figure, in the combination treatment group (LErafAON + Gemzar) compared to the single agent treatment group (LErafAON and Gemzar) or the control group (NS normal saline, BL, blank liposome) Growth inhibition was observed. Values shown are mean ± s. d. It is. As shown in FIG. 26B, the antitumor activity of the administered chemotherapeutic agents (Gemzar and Gencitabine HCl) was increased again as a result of the oligo / cationic liposome treatment.

Conclusion The results of Examples 4-8 clearly reveal that the effects of chemotherapy, particularly treatment of tumors resistant to chemotherapy, are included in the cationic liposome formulation of the present invention, such as genes expressed by tumors such as raf It has been proved that it can be enhanced by administration of oligos targeting the oncogenes of Since this efficacy has been shown for a variety of different chemotherapeutic agents, alone or in combination, as well as for different cancers, this enhancement has been shown for a variety of different cancers, particularly cancers that are resistant to chemotherapy or radiation therapy, and It is reasonable to expect to be observed with a variety of different chemotherapeutic agents alone or in combination.

Detailed description of the drawings
Liposome embedding of ATG-AS raf ODN. 5'-fluorescein labeled ATG-AS raf ODN was embedded in liposomes (PC / CHOL / DDAB) as described in the experimental method. Figure 2 shows bright field microscopy of blank liposomes (a) and fluorescence microscopy of liposomes containing blank liposomes (b) and fluorescein-labeled ATG-AS raf ODN (C-H). (A) Time course of cellular uptake of LE-ATG-AS raf ODN. SQ-20B cells were incubated with ³² P-5 ′ end labeled LE-ATG-AS raf ODN and excess unlabeled LE-ATG-AS raf ODN (10 μM) as described in Experimental Methods. Results are the mean ± sd from two independent experiments, each done in duplicate. d. (B) Intracellular stability of LE-ATG-AS raf ODN. Cells were incubated for 1 hour with ³² P-end labeled LE-ATG-AS raf ODN and excess unlabeled LE-ATG-AS raf ODN (10 μM in 1% FBS containing medium), washed with PBS, then Incubation was continued in medium containing 20% FBS for 0 hours (lane 2), 2 hours (lane 3), 8 hours (lane 4), 12 hours (lane 5), 24 hours (lane 6). Cells were lysed and ODN in various samples was analyzed by denaturing gel electrophoresis as described in Experimental Methods. Lane 2, radiolabeled control LE-ATG-AS raf ODN; 15mer, ATG-AS raf ODN; migration of xylene cyanol (XC) and bromophenol blue (BP). Plasma concentration-time profile of LE-ATC-AS raf ODN. 30 mg / kg LE-ATG-AS raf ODN (top panel) or TG-AS raf ODN (middle panel) were administered i.p. to Balb / dnu / nu mice. e. Administered. As described in the experimental methods, blood samples were collected from the retroorbital sinus at the indicated times after injection, and the ODN in the plasma samples was extracted and analyzed by denaturing gel electrophoresis. Standard prepared by spiking a known amount of ATG-AS raf ODN into St blank plasma. Top panel: Samples were diluted before electrophoresis as follows: lanes 1, 12 times; lanes 2 and 3, 4 times; lanes 4 and 5, 3.3 times; lanes 6, 2 times; 1.4 times; Lane 8, 1.3 times; Lane 9, 1 time. St lanes show 0.25, 0.5 and 1.0 μg / ml of ATG-AS raf ODN. 1.0 μg / ml of the standard sample corresponds to 18.4 μg of ODN. An additional standard (log range greater than 1.4-2) was used to measure ODN concentrations over a 24 hour period (data not shown). Middle panel: Samples were diluted before loading as follows: lanes 1, 4 times; lines 2 and 3, 2 times; lanes 4 and 5 and 6, 1 times; lane 7, 0.75 times; 8 and 0, 0.6 times. The St lane shows 0.125, 0.25, 0.5 μg / ml of ATG-AS raf ODN, and 0.5 μg / ml of the standard sample corresponds to 6.9 ng of ODN. Bottom panel: LE-ATG-AS raf ODN plasma concentration-time curve shown in the top panel. Quantification data was calculated based on comparison to a standard sample of known concentration and then normalization to the sample dilution factor used for loading. Tissue distribution profile of LE-ATG-AS raf ODN. Tissue samples were treated with 30 mg / kg LE-ATG-AS raf ODN i. v. Collected at the indicated times after dosing. As described in the experimental method, ODN was extracted from homogenized tissue and probed with ³² P-labeled ATG-S raf ODN. (A) Representative autoradiograph from liver and kidney, (b) 30 mg / kg LE-ATG-AS raf ODN dose i. v. ATG-AS raf ODN concentration in different tissues at the indicated time after administration. Quantification data was calculated based on comparison to a standard sample of known concentration and then normalization to the weight of the collected organ. Specificity of inhibition of Raf-1 protein expression by LE-ATG-AS rad ODN. (A) Time course analysis. Logarithmically growing SQ-20B cells were treated with 10% LE-ATG-S raf ODN (AS) or LE-ATG-S rad ODN (S) in medium containing 1% FBS for the indicated times. Untreated control cells (C) or cells treated with blank liposomes (10 μm) were simultaneously switched to 1% FBS containing medium for 8 hours. Whole cell lysates were normalized for total protein content and immunoprecipitated with agarose-conjugated polyclonal anti-Raf-1 antibody (Santa Cruz). Immune complexes were immunoblotted with polyclonal anti-RAF-1 antibody (top) as described in the experimental method. Results from three independent experiments are quantified and data are expressed against the level of Raf-1 in LE-ATG-S raf ODN treated cells (bottom). (B) Dose-response analysis. Logarithmically growing SQ-20B tumor cells were treated with medium containing 1% FBS for 8 hours with the indicated concentrations of LE-ATG-AS raf ODN (AS) or LE-ATG-S raf ODN (S). . Normalized cell lysates were analyzed for RAF-1 expression (top). Quantification data from three independent experiments is expressed against the level of Raf-1 in LE-ATG-S raf ODN treated cells (bottom). Inhibition of Raf-1 protein kinase activity by LE-ATG-AS raf ODN. Logarithmically growing SQ-20B cells were treated with 1% FBS-containing medium for 8 hours with 120 μm LE-ATG-AS raf ODN (AS) or 10 μm LE-ATG-S raf ODN (S). Control cells (C) were simultaneously switched to 1% FBS containing medium for 8 hours. Whole cell lysates were normalized for protein content and Raf-1 phosphotransferase activity was assayed in vitro using its physiological substrate, MKK1, as described in Experimental Methods. Radiolabeled reaction products were separated by electrophoresis and autoradiographed (inset). Quantification data from two independent experiments, each done in duplicate, are expressed as Raf-1 enzyme activity in LE-ATG-AS raf ODN treated cells versus LE-ATG-S raf ODN treated cells . Effect of intravenous administration of (our) LE-ATG-AS raf ODN on Raf-1 expression in normal and tumor tissues. Raf-1 expression was administered i.v. at a daily dose of 6 mg / kg of LE-ATG-AS raf ODN (AS) or LE-ATC-S raf ODN (S) for 5 consecutive days. v. Following injection, Balb / c nu / nu mouse liver, kidney, and SQ-20B tumor xenografts were tested. Control mice (C) received normal saline. Representative data showing Raf-1 expression in lysates normalized for protein content by immunoprecipitation and immunoblotting (top). Quantification data were obtained as mean ± sd from 3 mice. Shown as d (bottom). Inhibition of Raf-1 protein expression in SQ-20B tumor xenografts by intratumoral administration of LE-ATG-AS raf ODN: As described in the experimental method, each animal received 4 mg / kg daily for 7 days. Intratumoral injection of LE-ATG-AS raf ODN (AS) on the right flank and LE-ATG-S raf ODN (S) on the left flank. Shown is Raf-1 expression in right (AS) and left (S) tumor xenografts from two representative animals (inset). The quantification data shown is the mean ± sd from 3 representative mice. d. It is. Antisense sequence-specific inhibition of Raf-1 expression in SQ-20B cells. (A) SQ-20B cells in logarithmic growth were treated with LE-5132 (lanes 3, 5, 10), 5132 (lanes 4 and 6) or LE with the indicated ODN concentrations as described in Materials and Methods. Treated with -10353 (lane 9). Control cells were untreated (lanes 1 and 7) or treated with 1 μM blank liposomes (BL) (lanes 2 and 8). Whole cell lysates were normalized for total protein content and immunoprecipitated with agarose-conjugated, polyclonal anti-Raf-1 antibody. Immune complexes were separated by 7.5% SDS-PAGE and immunoblotted with a polyclonal anti-Raf-1 antibody. (B) Data from three independent experiments are quantified and expressed relative to the level of Raf-1 in untreated cells. Effect of LE-5132 on clotting time. Normal human plasma is mixed with the indicated concentrations of LE-5132 or 5132 or left untreated and purified at 37 ° C. for 1 minute with APTT reagent (allergic acid activator) Brain sephaline extract). The coagulation reaction is initiated by adding calcium chloride and the time required to form a visible clot is recorded manually in seconds. Data represent mean = SD from 3 experiments. Plasma concentration-time profiles of LE-5132 and 5132; 30 mg / kg LE-5132 or 5132 were administered i.v. to Balb / c nu / nu mice. v. Administered. Blood samples were collected from the retroorbital sinus at the indicated times after injection and ODN in the plasma samples was extracted and analyzed by denaturing gel electrophoresis as described. S1, S2, S3, standards prepared by spiking blank plasma with a known amount of 5132. (A) Samples were diluted prior to electrophoresis as follows: lane 1.15 times; lanes 2, 10 times; lanes 3 and 4, 5 times; lanes 5, 1 times; 8 times. (B) Samples were diluted prior to electrophoresis as follows: lanes 1, 15 fold; lanes 2, 10 fold; lanes 3 and 4, 5 fold; lanes 5, 1 fold; 8 times. S1, S2 and S3 represent 5132 of 0.25, 0.5 and 1.0 μg / ml, respectively. 1.0 μg / ml of the standard sample corresponds to 20 ng of ODN. (C) LE-5132 and 5132 plasma concentration-time curves. Quantification data was calculated based on comparison to a standard sample of known concentration and then normalized to the sample dilution factor used for loading. Normal tissue pharmacokinetics of LE-5132 / 5132 as a function of area under the concentration-time curve (AUC). Tissue samples were collected between 0 and 48 hours after administration of 30 mg / kg LE-5132 or 5132 as in FIG. ODN was extracted from the homogenized tissue and probed with ( ³² P) -labeled sense raf ODN. Quantitative analysis was performed based on comparisons with standard samples of known concentrations and then normalized to the weight of the collected organs. Effect of LE-5132 on SW-20B tumor growth. SQ-20B tumor cells (2 × 10 ⁶ ) were administered to the left flank region of each male Balb / c nu / nu mouse a. c. Injected. 10-12 weeks old. Tumor xenografts were grown to an average tumor volume of 94 ± 6.4 mm ³ and the animals were randomly divided into two treatment groups. Day 0 represents the first day of treatment. Mice receive 6 mg / kg LE-5132 or blank liposomes (BL) i.d. once daily for the first 7 days. v. Followed by 6 additional doses every other day as indicated by the arrows. The animals were killed on the 30th. Data shown are mean ± SE of 5-7 animals per group. Effect of LE-5132 on Raf-1 protein levels in SQ-20B tumors. (A) Tumor-bearing mice were treated with LE-5132 (iv, 10 mg / kg) or IR (3.8 Gy / day) or both as described in the legend to FIG. Tumors representing various treatment groups were excised on the 7th (lanes 1 and 2) and 14th (lanes 3-8) of treatment. Raf-1 expression was detected by immunoprecipitation in tissue homogenates normalized for protein content followed by immunoblotting. Lane 1, untreated control; Lane 2 and (nd) 3, BL; Lane 4, IR; Lane 5 and 6, LE-5132; Lane 7 and 8, LE-5132 + IR. (B) Quantification data shown is mean ± SE from two animals. Effect of LE-5132 and ionizing radiation on SQ-20B tumor growth. (A) SQ-20B tumor xenografts were grown in male Balb / c nu / nu mice as described. Animals bearing an average tumor volume of 72.0 ± 4.3 mm ³ were randomly divided into 5 treatment groups. Day 0 indicates the first day of treatment. Mice were given the number of days indicated, LE-5132 10 mg / kg i. v. (LE-5132) Treated with 3.8 Gy ionizing radiation (IR) once a day, or a combination of these two treatments (LE-5132 + IR). Control groups received blank liposomes (BL) or were untreated (C). The animals were killed on day 45. Data shown are mean ± SE of 5-7 animals per group. (B) A multiple of the change in mean tumor volume in the different treatment groups on day 30. The data shown is the mean ± SE from two independent experiments. 5-7 animals per group per experiment. ⁼ P <0.001 Histopathology of SQ-20B tumor. Tumors were excised 24 hours after the final treatment. Shown are examples of histopathology of tumors treated with untreated tumors (A), LE-5132 (B), IR (C), or LE-5132 + IR (D). 250 times. In vitro results of dose-response uptake experiments with unlabeled antisense raf oligo (ATG-AS) in free (ATG-AJ ^C ) or liposome (DMTAP = PC = CHOL) embedded form (LE-ATG-AS) including. Includes results of time course uptake experiments with SQ-20B timer cells using free (ATG-AS) or liposome-embedded (LE-ATG-AS) raf oligonucleotides. Includes half of the stability experiments comparing the stability of raf oligonucleotides in free (ATG-AS) or liposome-embedded (LE-ATG-AS) μm. Includes the results of another stability experiment comparing the stability of free raf oligonucleotide (ATG-AS) or liposome-embedded (LE-ATG-AS). DMTAP: PC: CHOL liposomes i. v. Contains survival data for administered mice / CD2F1. Includes survival data from murine CD2F1 mice administered raf oligonucleotide (LE-ATG-AS) in DMTAP: PC: CHOL liposomes. Anti-tumor efficacy of LEraf Aon in combination with cisplatin in athymic nu / nu mice bearing human prostate cancer (PC-3) xenografts. Male athymic mice were inoculated subcutaneously (sc) with 5 × 10 ⁶ PC-3 cells in 0.2 ml phosphate buffered saline per animal. Until tumor volume is 60 mm ³ 100 mm ^3, the tumor growth was twice monitored weekly. The animals were randomly divided into the above 5 treatment groups (n = 8). Mice were treated intravenously at the indicated dose via the tail vein and tumor size was monitored twice a week. Values shown are mean ± s. d. It is. Anti-tumor efficacy of LEraf AON in combination with epirubicin in athymic nu / nu mice bearing human prostate cancer (PC-3) xenografts. Male athymic mice received 5 × 10 ⁶ PC-3 cells in 0.2 ml phosphate buffered saline per animal s. c. Vaccinated. Tumor growth until tumor volume is 60 mm ³ 100 mm ^3, were twice monitored weekly. The animals were randomly divided into the above 5 treatment groups (n = 6). Mice were treated intravenously with the indicated dose via the tail vein and tumor size was monitored twice a week. Values shown are mean ± s. d. It is. Anti-tumor efficacy of LEraf AON in combination with mitoxantrone in athymic nu / nu mice bearing human prostate cancer (PC-3) xenografts. Male athymic mice received 5 × 10 ⁶ PC-3 cells in 0.2 ml phosphate buffered saline per animal. c. Vaccinated. Tumor growth until tumor volume is 60 mm ³ 100 mm ^3, were twice monitored weekly. The animals were randomly divided into the above 5 treatment groups (n = 5). Mice were treated intravenously with the indicated dose via the tail vein and tumor size was monitored twice a week. Values shown are mean ± s. d. It is. Anti-combination of liposome-entrapped raf antisense oligodeoxyribonucleotide (LErafAON) and Gemzar (NDC0002 + 7501 + 01, Gencitabine HCl, Eli Lilly and Company, Indianapolis, IN) in athymic mice carrying human pancreatic cancer xenografts (Colo357) Tumor effectiveness. Athymic mice were inoculated with approximately 1.0 × 10 ⁶ Colo357 human pancreatic cancer cells. Tumor ^volume, was put up to the size of 50mm ³ ~100mm 3. Tumor-bearing mice were randomly divided into 5 treatment categories (n = 5). LErafAON formulations were prepared as previously described (Gokhale et al., Gene Therapy 4,1299-1299, 1997; and Gokhale et al., Manuscript in preparation). The anticancer drug Gemzar (200 mg vial) was purchased from the Oncology Pharmacy at Georgetown University Hospital and reconstituted in normal saline (12.5 mg / ml). LErafAON (25.0 mg / kg) or Gemzar (750 mg / kg) was injected intravenously on the days indicated. Tumor volume was measured twice a week and the tumor size (initial%) for the various treatment groups was plotted. As shown in this figure, significant tumor growth was observed in the combination treatment group (LErafAON + Gemzar) compared to the single agent treatment group (LErafAON and Gemzar) or the control group (NS normal saline, BL blank liposome). Blocking was recognized. Values shown are mean ± s. d. It is. Liposome-trapped raf antisense oligodeoxyribonucleotide (LErafAON) and Gemzar (NDC002 + 7501 + 01, Gencitabine HCl, Eli Lilly and Company, Indianapolis, In) in athymic mice carrying human pancreatic cancer xenografts (Aspc-1) Anti-tumor efficacy. Athymic mice were inoculated with approximately 2.5 × 10 ⁶ Aspc-1 human pancreatic cancer cells. Tumor ^volume, was put up to the size of 50mm ³ ~100mm 3. Tumor-bearing mice were randomly divided into 5 treatment categories (n = 5). The LErafAON formulation was prepared as previously described (Gokhale et al., Gene Therapy 4,1289-1299, 1997; and Gokhale et al., Manuscript in preparation). The anticancer drug Gemzar (200 mg vial) was purchased from the Oncology Pharmacy at Georgetown University Hospital and reconstituted in normal saline (12.5 mg / ml). LErafAON (25.0 mg / kg) or Gemzar (100.0 mg / kg) was injected intravenously on the days indicated. Tumor volume was measured twice a week and the tumor size (initial%) for the various treatment groups was plotted. As shown in this figure, in the combination treatment group (LErafAON + Gemzar) compared to the single drug treatment group (LErafAON) and Gemzar) or the control group (NS normal saline, BL blank liposome) Tumor growth inhibition was observed. Values shown are mean ±.

Claims (22)

  A method for chemical sensitization of tumor tissue, comprising a chemotherapeutic agent and a cationic liposome comprising a cationic lipid, phosphatidylcholine and cholesterol, in which at least one oligonucleotide is embedded Administering a product.   The method of claim 1, wherein the oligonucleotide is in the size range of 10-40 nucleotides and is phosphorothioated only at the terminal nucleotide.   The method of claim 1, wherein the oligonucleotide comprises 10 to 40 nucleotides and all of its bases are phosphorothioated.   The method of claim 1, wherein the oligonucleotide is of a size in the range of 10-40 nucleotides and all of its bases are modified in a chimeric form.   The method of claim 1, wherein the oligonucleotide is administered intravenously.   The method of claim 1, wherein the oligonucleotide is administered directly to the target tissue.   The method of claim 1, wherein the oligonucleotide is administered during arterial circulation toward the target tissue.   The composition of claim 1, wherein the oligonucleotide is antisense DNA.   The method of claim 1, wherein the oligonucleotide has the formula 5′-GTGCTCTCATTGATGC-3 ′ (SEQ ID NO: 1) and only the terminal base is phosphorothioated.   Consisting essentially of a cationic lipid such as dimethyldioctadecylammonium bromide (DDAB), phosphatidylcholine (PC), and cholesterol, the sequence 5′-GTGCTCCATTGATGC-3 ′ (SEQ ID NO: 1), only the terminal sequence phosphorothioated A composition of matter comprising a liposome comprising a sequence that has been prepared.   It consists essentially of a cationic lipid such as dimyristoyl trimethylammonium propane (DMTAP), phosphatidylcholine (PC) and cholesterol, and is of sequence 5′-GTGCTCCATTGATGC-3 ′ (SEQ ID NO: 1), wherein only the terminal sequence is phosphorothioated. A composition of matter comprising a liposome comprising a sequence.   2. The method of claim 1, wherein the chemotherapeutic agent is an alkylating agent, an antimetabolite, a natural product, a hormone or an antagonist.   The method according to claim 1, wherein the chemotherapeutic agent is a platinum coordination complex, anthracenedione, substituted urea, methylhydrazine derivative, adrenocortical inhibitor, small molecule inhibitor, peptide, antibody or tyrosine kinase inhibitor.   The method of claim 1, wherein the chemotherapeutic agent is epirubicin, doxorubicin, docetaxel, or paclitaxel.   The method of claim 1, wherein the chemotherapeutic agent is cisplatin or mitoxantrone.   The method of claim 1, wherein the chemotherapeutic agent is gencitabine.   2. The method of claim 1, wherein the cancer is leukemia, lymphoma, myeloma, carcinoma, or sarcoma.   2. The method of claim 1, wherein the oligonucleotide is administered before or after the chemotherapeutic agent.   2. The method of claim 1, wherein several different oligonucleotides are administered before or after the chemotherapeutic agent.   The method of claim 1, wherein the oligonucleotide is administered before or after more than one chemotherapeutic agent.   2. The method of claim 1, wherein the oligonucleotide is administered before or after the combination of radiation and chemotherapeutic agent.   2. The method of claim 1, wherein more than one oligonucleotide is administered before or after the combination of radiation and chemotherapeutic agent.